Acceleration and Automation of Solid Sample Treatment, Volume 24 (Techniques and Instrumentation in Analytical Chemistry)

TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY- VOLUME 24 ACCELERATION AND AUTOMATION OF SOLID SAMPLE TREATMEN...

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TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY- VOLUME 24

ACCELERATION AND AUTOMATION OF SOLID SAMPLE TREATMENT

TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY Volume 1 Volume 2 Volume 3 Volume 4

Volume 5 Volume 6 Volume 7 Volume 8 Volume 9 Volume 10 Volume 11 Volume 12 Volume 13 Volume 14 Volume 15 Volume 16 Volume 17 Volume 18 Volume 19 Volume 20 Volume 21 Volume 22 Volume 23 Volume 24

Evaluation and Optimization of Laboratory Methods and Analytical Procedures. A Survey of Statistical and Mathematical Techniques by D.L. Massart, A. Dijkstra and L. Kaufman Handbook of Laboratory Distillation by E. Krell Pyrolysis Mass Spectrometry of Recent and Fossil Biomaterials. Compendium and Atlas by H.L.C. Meuzelaar, J. Haverkamp and F.D. Hileman Evaluation of Analytical Methods in Biological Systems Part A. Analysis of Biogenic Amines edited by G.B. Baker and R.T. Coutts Part B. Hazardous Metals in Human Toxicology edited by A. Vercruysse Part C. Determination of Beta-Blockers in Biological Material edited by V. Marko Atomic Absorption Spectrometry edited by J.E. Cantle Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry by D.M. Desiderio Electroanalysis. Theory and Applications in Aqueous and Non-Aqueous Media and in Automated Chemical Control by E.A.M.F. Dahmen Nuclear Analytical Techniques in Medicine edited by R. Cesareo Automatic Methods of Analysis by M. Valcarcel and M.D. Luque de Castro Flow Injection Analysis- A Practical Guide by B. Karlberg and G.E. Pacey Biosensors by F. Scheller and F. Schubert Hazardous Metals in the Environment edited by M. Stoeppler Environmental Analysis. Techniques, Applications and Quality Assurance edited by D. Barcel6 Analytical Applications of Circular Dichroism edited by N. Purdie and H. Brittain Trace Element Analysis in Biological Specimens edited by R.F.M. Herber and M. Stoeppler Flow-through (Bio)Chemical Sensors by M. Valcarcel and M.D. Luque de Castro Quality Assurance for Environmental Analysis edited by Ph. Quevauviller, E.A. Maier and B. Griepink Instrumental Methods in Food Analysis edited by J.R.J. Pare and N.M.R. B(~langer Trace Determination of Pesticides and their Degradation Products in Water by D. Barcel6 and M.-C. Hennion Analytical Pyrolysis of Natural Organic Polymers by S.C. Moldoveanu Sample Handling and Trace Analysis of Pollutants edited by D. Barcelo Interlaboratory Studies and Certified Reference Materials for Environmental Analysis: the BCR approach by Ph. Quevauviller and E.A. Maier Molecularly Imprinted Polymers Man-made mimics of antibodies and their applications in analytical chemistry edited by B. Sellergren Acceleration and Automation of Solid Sample Treatment M.D. Luque de Castro and J.L. Luque Garcfa

TECHNIQUES AND INSTRUMENTATION IN ANALYTICAL CHEMISTRY- VOLUME 24

ACCELERATION AND AUTOMATION OF SOLID SAMPLE TREATMENT Maria Dolores Luque de Castro Jose Luis Luque Garcia Department of Analytical Chemistry, University of Cordoba Campus of Rabanales, 14071 Csrdoba, Spain

2002

ELSEVIER Amsterdam - B o s t o n - L o n d o n - N e w S a n Diego - S a n F r a n c i s c o - S i n g a p o r e

York - Oxford - Sydney

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Paris

- Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 RO. Box 211, 1000 AE Amsterdam, The Netherlands 92002 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single pohtocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier Science via their homepage (http://www. elsevier.com) by selecting 'Customer support' and then 'Permissions'. Alternatively you can send an email to: [email protected], or fax to: (+44) 1865 853333. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+1) (978) 7508400; fax: (+1) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WlP 0LP, UK; phone (+44) 207 631 5555; fax: (144) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Global Rights Department, at the mail, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2002 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. ISBN: 0-444-50716-7 Typeset by Q3 Bookwork, United Kingdom @ The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The Netherlands.

Contents

Foreword by Freddy Adams ................................................................................... Preface ....................................................................................................................

C H A P T E R 1.

xiii XV

I N T R O D U C T I O N TO SOLID S A M P L E PRETREATMENT

1.1

Conventional procedures for sample pretreatment and their standardization ............................................................................................ 1.2 The state o f the art in sample preparation ................................................. 1.3 Problems encountered in automating sample preparation ......................... 1.4 Batch versus serial approaches to automated sample preparation ............ 1.5 Definitions and restrictions o f term meanings ........................................... 1.5.1 Sample preparation and sample pretreatment ................................ 1.5.2 Solid sampling ................................................................................ 1.5.3 Digestion versus leaching ............................................................... 1.5.4 Use o f a leaching step versus a digestion step .............................. 1.5.5 Leaching versus extraction ............................................................. 1.6 Some notes on the book contents ............................................................... References

C H A P T E R 2. 2.1 2.2

2.3 2.4

2.5

1 2 4 5 7 7 7 8 8 8 9 10

ANALYTICAL FREEZE-DRYING

Introduction ................................................................................................. Steps o f the freeze-drying process ............................................................. 2.2.1 The freezing step ............................................................................ 2.2.2 The sublimation step ....................................................................... 2.2.3 The desorption step ........................................................................ Variables influencing the freeze-drying process ........................................ Freeze-drying methods ............................................................................... 2.4.1 The manifold method ..................................................................... 2.4.2 The batch method ........................................................................... 2.4.3 The bulk method ............................................................................. Computer-assisted analytical freeze-drying ............................................... 2.5.1 Computer-assisted freezing ............................................................. 2.5.2 Computer-assisted sublimation .......................................................

11 12 12 13 16 17 19 19 21 21 21 21 22

vi

Contents

2.5.3 Computer-assisted desorption ......................................................... Laboratory-scale freeze-dryers ................................................................... 2.6.1 Basic components of a laboratory freeze-dryer ............................. 2.6.2 Evolution of laboratory freeze-dryers ............................................ 2.6.3 Laboratory-designed freeze-dryers ................................................. 2.6.4 Commercially available freeze-drying equipment ......................... General aims of analytical freeze-drying ................................................... 2.7 2.7.1 Increasing long-term storage stability ............................................ 2.7.2 Preparing tissues for microscopic examination .............................. 2.7.3 Preparing samples as an intermediate step in (bio)chemical analytical procedures ...................................................................... 2.7.4 Recovering products from a reaction mixture ................................ Analytical uses of freeze-drying ................................................................. 2.8 2.8.1 Reagents .......................................................................................... 2.8.2 Standards ......................................................................................... 2.8.3 Samples ........................................................................................... References

2.6

CHAPTER 3.

Introduction ................................................................................................. General aspects of the cavitation phenomenon .......................................... Types of ultrasonic devices ......................................................................... Ultrasound-assisted leaching ...................................................................... 3.4.1 Batch systems ................................................................................. 3.4.2 Continuous systems ........................................................................ 3.4.3 Ultrasound-assisted leaching versus other leaching techniques .... 3.5 Ultrasound-assisted sampling ..................................................................... 3.5.1 Ultrasonic nebulizers ...................................................................... 3.5.2 Ultrasonic slurry sampling ............................................................. 3.5.3 Acoustically levitated droplets ....................................................... 3.6 Ultrasound-assisted electroanalysis ............................................................ 3.7 Other analytical uses of ultrasounds .......................................................... 3.7.1 Ultrasonic degassing ................................................................................... 3.7.2 Ultrasonic filtration ..................................................................................... References ..........................................................................................................

4.1 4.2

31 32 32 33 33 35 39

A N A L Y T I C A L U S E S OF ULTRASOUNDS

3.1 3.2 3.3 3.4

CHAPTER 4.

23 23 23 24 26 27 30 30 31

43 44 46 48 49 54 60 61 62 66 69 71 75 75 77 79

SOLID SAMPLE TREATMENTS INVOLVING THE REMOVAL OF VOLATILE SPECIES

Introduction ................................................................................................. Hydride and cold mercury vapour generation ........................................... 4.2.1 Introduction ..................................................................................... 4.2.2 Samples, analytes and reagents ...................................................... 4.2.3 The separator and its associated equipment ................................... 4.2.4 Variables influencing vapour generation ........................................

83 84 84 84 86 89

Contents

Features of methods based on hydride or cold mercury vapour generation ........................................................................................ 4.2.6 Applications .................................................................................... 4.3 Headspace sampling ................................................................................... 4.3.1 Introduction ..................................................................................... 4.3.2 Equipment and procedures ............................................................. 4.3.3 Theoretical background .................................................................. 4.3.4 Variables affecting performance ..................................................... 4.3.5 Calibration and analytical features ................................................. 4.3.6 Applications .................................................................................... 4.4 Analytical pervaporation ............................................................................. 4.4.1 Introduction ..................................................................................... 4.4.2 Principles o f p e r v a p o r a t i o n ............................................................. 4.4.3 The analytical pervaporator and auxiliary units ............................ 4.4.4 Pervaporation efficiency .................................................................. 4.4.5 Variables influencing analytical pervaporation .............................. 4.4.6 The physics of analytical pervaporation: kinetics of the mass transfer process ...................................................................... 4.4.7 Analytical features o f p e r v a p o r a t i o n methods ............................... 4.4.8 Scope of application of analytical pervaporation ........................... 4.4.9 Prospects for analytical pervaporation ........................................... 4.5 Solid-phase microextraction (SPME) ......................................................... 4.5.1 Introduction ..................................................................................... 4.5.2 Principles, devices and theoretical aspects of SPME .................... 4.5.3 Variables affecting SPME ............................................................... 4.5.4 Analytical features of SPME .......................................................... 4.5.5 Advantages and shortcomings of SPME ........................................ 4.5.6 Applications of SPME .................................................................... References

vii

4.2.5

CHAPTER 5. 5.1 5.2

5.3

5.4

90 91 93 93 93 103 113 121 125 128 128 128 130 134 135 139 141 143 154 154 154 155 161 169 170 171 173

MICROWAVE-ASSISTED SOLID SAMPLE TREATMENT

Introduction ................................................................................................. Fundamentals of microwave energy and its interaction with matter ......... 5.2.1 Dissipation factor ............................................................................ 5.2.2 Transformation of microwave energy into heat ............................. 5.2.3 Microwave heating .......................................................................... Microwave equipment ................................................................................. 5.3.1 Main components of a microwave device ...................................... 5.3.2 Closed-vessel microwave systems .................................................. 5.3.3 Open-vessel microwave systems .................................................... 5.3.4 Closed versus open microwave systems ......................................... Variables governing microwave-assisted processes .................................... 5.4.1 Microwave power and exposure time .............................................

179 180 181 181 182 183 184 186 192 205 207 207

viii

Contents

5.4.2 5.4.3 5.4.4

Temperature and pressure ............................................................... Type o f solvent ................................................................................ Influence o f sample viscosity and sample size on microwave heating ............................................................................................. Applications o f microwaves to solid sample treatment ............................. 5.5 5.5.1 Microwave-assisted digestion ......................................................... 5.5.2 Microwave-assisted extraction ........................................................ 5.5.3 Microwave-assisted sample drying ................................................. 5.5.4 Microwave-assisted distillation ....................................................... 5.5.5 Microwave-assisted protein hydrolysis ........................................... Safety considerations on the use o f microwave energy ............................. 5.6 References

CHAPTER 6.

211 212 212 218 222 223 223 224 225

HIGH-PRESSURE, HIGH-TEMPERATURE SOLVENT EXTRACTION

Introduction ................................................................................................. Variables affecting the extraction process .................................................. 6.2.1 Temperature ..................................................................................... 6.2.2 Pressure ........................................................................................... 6.2.3 Solvent type .................................................................................... 6.2.4 Solvent volume ............................................................................... 6.2.5 Solvent flow-rate ............................................................................. 6.2.6 Matrix composition ......................................................................... 6.2.7 Sample size ..................................................................................... 6.2.8 Extraction time ................................................................................ Accelerated solvent extraction (ASE) ........................................................ 6.3 6.3.1 Steps involved in the ASE process ................................................. 6.3.2 ASE devices .................................................................................... 6.3.3 Combination o f ASE with other operations o f the analytical process ............................................................................................. 6.3.4 Applications of ASE ....................................................................... 6.3.5 Comparison o f ASE with other solid-liquid extraction (leaching) techniques ........................................................................................ Dynamic pressurized hot solvent extraction (DPHSE) .............................. 6.4 6.4.1 D P H S E devices ............................................................................... 6.4.2 Steps o f the D P H S E process .......................................................... 6.4.3 Water as a leaching agent ............................................................... 6.4.4 Automation and improvement o f steps subsequent to D P H S E ..... 6.4.5 Applications o f D P H S E .................................................................. 6.4.6 Comparison of D P H S E with other leaching techniques ................ References

6.1 6.2

208 208

233 235 236 237 238 239 240 240 241 242 242 243 245 247 249 253 259 260 263 265 266 269 273 274

ix

Contents

CHAPTER 7.

ANALYTICAL SUPERCRITICAL

FLUID

EXTRACTION 7.1 7.2

Introduction ................................................................................................. Properties of supercritical fluids ................................................................. 7.2.1 Properties of the supercritical region ............................................. 7.2.2 The solubility parameter ................................................................. 7.3 Laboratory-built and commercial supercritical fluid extractors ................ 7.3.1 Basic components o f a supercritical fluid extractor ....................... 7.3.2 Operational modes .......................................................................... 7.3.3 Commercial supercritical fluid extractors ...................................... 7.4 Variables influencing supercritical fluid extraction (SFE) ......................... 7.4.1 Properties of the supercritical fluid ................................................ 7.4.2 Properties of the analyte ................................................................. 7.4.3 Properties of the sample ................................................................. 7.4.4 Dynamic and geometric variables .................................................. 7.4.5 Analyte collection modes ............................................................... 7.5 Approaches to improving SFE performance .............................................. 7.5.1 Using an alternative supercritical fluid .......................................... 7.5.2 Ion-pairing ....................................................................................... 7.5.3 Esterification and related reactions ................................................ 7.5.4 Formation o f o r g a n o m e t a l s ............................................................. 7.5.5 Chelation ......................................................................................... 7.5.6 Micellization ................................................................................... 7.6 Hyphenated techniques ............................................................................... 7.6.1 Supercritical fluid extraction-chromatography .............................. 7.6.2 Miscellaneous combinations ........................................................... 7.6.3 Types of detectors used in combination with supercritical fluid extractors ................................................................................ 7.7 Applications of supercritical fluid extraction ............................................. 7.7.1 Selectivity in supercritical fluid extraction ..................................... 7.7.2 Scope of application ....................................................................... 7.7.3 Comparison with other analyte removal techniques ...................... 7.8 Present and future of supercritical fluid extraction .................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTER 8.

8.1 8.2

281 281 283 285 286 286 290 290 292 294 300 301 303 306 307 309 311 312 312 313 314 315 316 321 325 328 329 329 331 340 341

DEVICES FOR SOLID SAMPLE TREATMENT PRIOR TO INTRODUCTION INTO ATOMIC SPECTROMETERS: ELECTROTHERMAL DEVICES AND GLOW-DISCHARGE SOURCES

Introduction ................................................................................................. Electrothermal atomizers and vaporizers ................................................... 8.2.1 Fundamentals of electrothermal vaporizers and atomizers ............ 8.2.2 Solid sampling modes in electrothermal vaporizers and atomizers .........................................................................................

347 348 348 355

x

Contents

8.2.3

Variables of solid sampling with electrothermal vaporizers and atomizers .................................................................................. 8.2.4 Steps of an electrothermal solid sampling process ........................ 8.2.5 Instrument parameters affecting solid sampling with electrothermal atomizers and vaporizers ........................................ 8.2.6 Use of modifiers in electrothermal solid sampling ........................ 8.2.7 Shortcomings of electrothermal sampling ..................................... 8.2.8 Calibration in methods involving electrothermal sampling of solid samples and slurries ............................................................... 8.2.9 Applications of electrothermal solid sampling prior to introduction into an AAS, ICP-AES, AFS or ICP-MS instrument ....................................................................................... 8.3 Glow-discharge sampling ........................................................................... 8.3.1 Introduction ..................................................................................... 8.3.2 Principles of the glow-discharge .................................................... 8.3.3 Glow-discharge sources: geometry and improvements .................. 8.3.4 Nature of the sample for glow-discharge sampling ....................... 8.3.5 Sample state and preparation for glow-discharge sampling .......... 8.3.6 Variables affecting glow-discharge sampling ................................. 8.3.7 Glow-discharge sampling as coupled to spectrometric detection... 8.3.8 Advantages and restrictions of glow-discharge sampling .............. 8.3.9 Models and calibration in methods involving glow-discharge sampling .......................................................................................... 8.3.10 Applications of glow-discharge sampling in combination with spectrometries ................................................................................. 8.3.11 Trends in glow-discharge sampling ................................................ 8.4 Other solid sampling approaches ............................................................... 8.4.1 Arc nebulization .............................................................................. 8.4.2 Direct sampling insertion devices .................................................. References

CHAPTER 9. 9.1 9.2

358 364 366 366 373 374

377 385 385 386 393 399 399 400 404 413 413 416 424 425 425 427 427

LASER-ASSISTED SOLID SAMPLING

Introduction ................................................................................................. Laser ablation .............................................................................................. 9.2.1 Features of ablation lasers .............................................................. 9.2.2 Steps and thresholds in laser ablation ............................................ 9.2.3 Craters and amount of ablated material ......................................... 9.2.4 Sample preparation for laser ablation ............................................ 9.2.5 Ablation cells .................................................................................. 9.2.6 Transport of ablated material .......................................................... 9.2.7 Mixed gas sample introduction ...................................................... 9.2.8 Equipment ....................................................................................... 9.2.9 Calibration techniques for laser ablation-atomization-ionizationexcitation-detection ........................................................................

435 437 437 440 441 442 443 445 445 446 446

Contents

9.2.10 Shortcomings of laser ablation ....................................................... 9.2.11 Selected applications of laser ablation sampling prior to atomization-ionization-excitation-detection ................................. 9.2.12 Comparison of laser-assisted sampling with other sampling techniques ........................................................................................ Laser-induced plasma spectroscopy ........................................................... 9.3 9.3.1 Introduction ..................................................................................... 9.3.2 Features of LIBS ............................................................................. 9.3.3 Steps and thresholds in LIBS ......................................................... 9.3.4 Experimental set-up for LIBS ........................................................ 9.3.5 Basic aspects of LIBS .................................................................... 9.3.6 Variables affecting LIBS performance ........................................... 9.3.7 Shortcomings of LIBS .................................................................... 9.3.8 Data collection: detector delay ....................................................... 9.3.9 Data analysis ................................................................................... 9.3.10 Applications of LIBS ...................................................................... 9.3.11 Comparison of LIBS with alternative techniques .......................... 9.3.12 Laser-induced breakdown-mass spectrometry ............................... References

CHAPTER 10.

xi 448 449 456 461 461 461 462 464 464 466 473 477 479 480 489 492 495

ROBOTIC SOLID SAMPLE PRETREATMENT

10.1 10.2

Introduction ................................................................................................. Workstations, robots, modules and peripherals .......................................... 10.2.1 Workstations .................................................................................... 10.2.2 Robots ............................................................................................. 10.2.3 Modules and peripherals ................................................................. 10.2.4 Detectors ......................................................................................... 10.2.5 Miscellaneous considerations on workstations and robotic stations ............................................................................................ 10.3 The role of robots in the analytical process ............................................... 10.3.1 Single-task robots and simple uses of robotics .............................. 10.3.2 Sample preparation ......................................................................... 10.3.3 Robotic development of the whole analytical process ................... 10.3.4 Combining robotic and continuous systems for more reliable development of the whole analytical process ................................. 10.4 Analytical scope of application of robotics ............................................... 10.5 Present and future of robotics .................................................................... References ..............................................................................................................

501 503 503 506 508 510

Index ......................................................................................................................

529

511 512 512 513 515 516 521 524 526

This Page Intentionally Left Blank

Foreword Writing a book is always an audacious act, but writing a book such as this one on sampling and sample pretreatment is even more than that: it is a real challenge. The book encompasses the entire field of analytical chemistry and oozes a profound knowledge of the concepts involved. The role of the analytical chemist has changed profoundly over the past few decades. The levels of analytes that can be determined routinely have decreased by several orders of magnitude and the sample sizes that need to be handled have dropped significantly in recent years. In parallel, the overall number of determinations that need to be performed has grown enormously. Also, the advent of intelligent instruments and laboratory automation has help analytical chemistry become a real information science. This significant, steady progress in analytical science, however, has been brought about as much by gradual advances in the preliminary steps of the analytical process (e.g. sample handling) as by a number of leaps in technological progress. Often, in discussing analytical science, chemical measurements are reduced to fundamental processes such as detection, identification or quantitation. Although analytical and data processing techniques are dealt with in many books, sampling and sample pretreatment have so far been the subject matters of only a few books and review articles. This sustained lack of attention is surprising if one considers the overall time spent on these preparatory stages leading to the final analytical act and when one realizes how intricately the quality of the results depends on the initial, preparatory steps of an analysis. The development of clean sampling and handling techniques, which involves substantial investments of time and resources, has always been a mandatory step towards obtaining accurate, reliable final analytical data. Scientific journals are rarely considered to be appropriate forums for discussing the guiding principles behind sampling and sample pretreatment. A book like this, which brings together all relevant wisdom and knowledge on this particular field, is therefore a more than welcome addition to the analytical chemist's library. This particular book is designed to provide the analytical chemist with the background required to understand the concepts inherent in automated solid sample pretreatment. Its chapters provide a logical, systematic introduction to all aspects involved in the different methods of sample pretreatment and their automation. Topics such as freeze-drying, ultrasounds, microwave treatment, high pressure, high temperature solvent extraction, superfluid extraction, laser ablation and laser-induced plasma spectroscopy, the use of electrothermal devices and glow-discharge processes, are dealt with in sequence in all their relevant aspects. One chapter is devoted to specific sample pretreatment methods

xiv

Foreword

involving the removal of volatile species and provides a comprehensive discussion of hydride generation, headspace sampling, pervaporation and solid-phase micro-extraction. Automated and computer-controlled methods are given proper emphasis throughout, and the role of workstations and robots in the analytical process is discussed in a separate chapter. Many of the ideas presented in this book have matured through the years from the wide experience gathered by the authors in the pursuit of their research on analytical chemistry and practical analysis at the University of C6rdoba, where Professor Luque de Castro leads a research group specializing in "Innovations in Continuous and Discrete (Robotic) Systems for the Automation of Analytical Methods" at the Department of Analytical Chemistry. Her research interests include the combined use of discrete (robotic) and continuous (flow injection and completely continuous flow) systems for the development of fully automated methods; the design of flow-through (bio)chemical sensors; immunoassays; chromatographic and non-chromatographic continuous separation techniques; solid-sample pretreatment by subcritical and supercritical fluid extraction; focused and multimode microwaves and ultrasounds; laser-induced processes; applications to environmental, clinical and food analysis; and on-line process monitoring. She has co-authored several books on subjects such as Flow Injection Analysis, Automatic Methods of Analysis, Non-Chromatographic Continuous Separation Techniques, Analytical Supercritical Fluid Extraction and Flow-Through (Bio)chemical Sensors. Freddy Adams University of Antwerp, Belgium

Preface Analytical Chemistry has traditionally been largely ignored by society and even by the scientific community, which, at most, has used it in the service of the other sciences. At present, Chemistry itself appears to have been reduced to a similar status. For example, the cover story of the August 20, 2001 issue of Time magazine was entitled "America's Best Science and Medicine" and, in it, the editors focused on the most exciting fields of research and then looked for the men and women who were doing the most cutting-edge research in such fields. The eighteen individuals honoured by the magazine none of whom was a chemist were conducting work in fields such as cell biology, the human origin, child psychology, pediatrics, genomics, cardiology, oncology, climatology, ecology, AIDS research, astrophysics, paleontology, biomedical engineering, neurobiology, cell death, spinal cord repair and molecular mechanics. In this issue, Time reflected present social perceptions in positing the following two destructive equations: science = biology and cutting edge = medical progress. Clearly, however, all these sciences rely on Chemistry and, ultimately, on Analytical Chemistry - - which, invariably, provided the data that led to the achievements distinguished by Time. We analytical chemists have always been aware of such unfair ignorance and yet have continued to work in pursuance of our goals. This book is a modest contribution intended to help those aiming to meet one of the most pressing needs of Analytical Chemistry: automation. A need that has arisen from the increasing demand for analyses in various fields of social interest including industry, health and the environment. Because sample pretreatment is the single step of the analytical process in greatest need of automation, this book compiles the wide range of tools available for this purpose with a view to assisting current and future users in choosing the best tool for each problem. Also, because full automation is almost always impossible, the primary goal and also the main achievement in some c a s e s - is to expedite this analytical step. Hence the book's title. Rather than a comprehensive discussion of available choices for expediting or automating sample pretreatment, this book provides a description of the most widely favoured choices at present and those with a high potential which, however, remains largely unexplored. Its ten chapters revolve around this criterion. The first chapter introduces both general aspects of sample preparation and the main problems encountered in automating sample treatments. The second provides a brief discussion of the underexploited potential of freeze-drying for delivering samples in forms that facilitate their subsequent analysis. Chapter 3 is devoted to a kind of energy that has also received inadequate attention from analysts: ultrasounds.

xvi

Preface

Those analytes that are either volatile or easily converted into volatile products are good candidates for separation from solid matrices by use of classical techniques such as hydride generation and dynamic or static headspace sampling, or more recent alternatives such as solid-phase microextraction and analytical pervaporation with which virtually the whole research group headed by the authors is acquainted; all are dealt with in Chapter 4. The growing use of microwaves in both batch and continuous systems is the subject matter of Chapter 5, where the authors have strived whether successfully or not readers will tell to avoid overemphasizing their own work. The controversial, sparse applications of high-pressure, high-temperature solvent extraction, a widely used technique of a high potential, are systematically discussed in Chapter 6. Chapter 7 describes the rapid expansion of supercritical fluid extraction but also how its scope has been restricted by differences in the behaviour of analytes naturally occurring in samples and those added to them for the purpose of analysis. Chapters 8 and 9 deal with various atomic techniques in which the authors are no experts, so any omissions or overpraising are totally unintentional. Chapter 8 is concerned with the use of electrothermal vaporizers and atomizers, and glow-discharge sources, with solid samples; it also provides a brief description of alternative techniques with a weaker impact that include arc nebulization and direct solid sample insertion. Chapter 9 describes the most salient laser-based techniques for solid sampling, namely: laser ablation and laser-induced breakdown spectroscopy; this chapter and the previous one examine the advantages and disadvantages of coupling the respective devices to analytical instruments. Finally, Chapter 10 focuses on robotic treatment, the only currently available choice for completely automated solid sampling by virtue of its ability to have samples weighed without human intervention. Because of their high cost, robotic stations and, especially, workstations, are inaccessible to many users, so their capabilities are only outlined in the book. Would-be users should thus seek more comprehensive information which continues to grow and change day by day, particularly as regards workstations before they embark on the costly venture of setting up a robotic configuration. The authors wish to express their gratitude to Antonio Losada, MSc, for his linguistic revision of the manuscript, Francisco Doctor for drawing the artwork and Jos6 Manuel Membrives for producing the tables. The authors C6rdoba, December 2001

Chapter 1

Introduction to solid sample pretreatment 1.1. CONVENTIONAL PROCEDURES FOR SAMPLE PRETREATMENT AND THEIR STANDARDIZATION

Analytical processes typically consist of several discrete steps. While some steps such as measurement and transduction of the instrumental signal, and data collection and processing, are common to all analytical processes, those preceding the insertion of the treated sample into the instrument for measurement (viz. sampling, dissolution, cleanup, preconcentration, individual separation, derivatization), which are critical with a view to ensuring the obtainment of accurate, reproducible results, vary markedly from process to process. Virtually all analytical methods include a sample preparation step. Despite the dramatic advances in analytical equipment and microcomputer technology, many sample preparation practices continue to rely on 19th-century technologies. For example, the ubiquitous Soxhlet extraction was developed more than one hundred years ago; also, many of the sample preparation techniques currently in use have been around for decades with little or no improvement over the years. The most common methods for analyte removal or sample dissolution are as follows: (a) Leaching, which involves a solid-liquid extraction with an appropriate solvent (water for ionic compounds or a more or less polar solvent for non-ionic substances). (b) Sample digestion, which is usually done with pure or mixed concentrated acids. (c) Fusion, in which the sample is mixed with an appropriate flux material and heated until melting, after which the mixture is allowed to cool and the fusion cake dissolved in a suitable solvent. (d) Dry ashing, which is especially common as a preliminary step in the analysis of organic samples for metallic and non-metallic elements. The sample is heated at 400-500~ in a muffle furnace for several hours and the target elements are either converted into gaseous species and collected into an appropriate absorber for determination or kept as a residue and dissolved into a suitable acid for analysis. One of the main constraints on conventional procedures for sample preparation is their lack of uniformity. In fact, many users introduce specific modifications in the general procedures that eventually result in enormous differences in the way they are implemented. Such a lack of uniformity precludes comparison of results obtained using what was seemingly the same procedure and validation of the ensuing methods. Comparability of the results can thus only be achieved by using actually identical procedures; this justifies the efforts currently in progress at standardizing sample preparation procedures.

2

Introduction to solid sample pretreatment

While some authors argue that standardization may "fossilize" progress in analytical science in a wide range of cases, it is widely accepted that the only way to ensure comparability when using operationally defined procedures is to standardize such procedures and apply them according to very strict protocols. In response to this need, the Standards, Measurements and Testing (SM&T) programme (formerly BCR) of the European Union developed a number of protocols for standardizing procedures over the past two decades, prominent examples of which are the operationally defined extraction procedures for soil and sediment analysis [1]. Standardizing a given procedure involves the following actions:

(a) Feasibility studies or examination of the literature, consultation with experts and selection of a few methods. (b) lnterlaboratory studies using the selected methods and reference materials. From the results, changes in the method to be used in the last step may be decided upon. (c) Validation through interlaboratory studies, in which the participating laboratories receive natural samples for analysis following a strict protocol. The results thus obtained lead to either adopting the method or restarting the standardization process by reselecting methods in the light of the outcome of the failed attempt. Standardization studies are slowly being superseded by sample preparation equipment that comes with bundled cookbooks where the optimum operating conditions for each procedure are clearly described. Some time is required in most cases, however, for universal adoption of automated procedures following extensive application by users.

1.2. THE STATE OF THE ART IN SAMPLE PREPARATION Usually, even simplest types of samples are unsuitable for direct analysis; in most cases, they are too dilute, too concentrated or simply incompatible with normal instrument operation. The solution to these and other problems has been sample preparation using a wide range of approaches many of which, as noted in the previous section, have been in use for decades. Sample preparation (SP) remains one of the more timeconsuming, error-prone aspects of analytical chemistry, so much so that it is increasingly being recognized as a legitimate area of specialization in this science [2]. In fact, SP technology is gaining increasing momentum with the rapid development of specialized equipment, much in the same manner as was formerly the case with chromatography and spectroscopy. While the last two are by now mature, well-established techniques that are even recognized as analytical subdisciplines, sample preparation is still consolidating, even though it is expanding and growing at an incredible rate. Thus, SP has rapidly emerged as a specialized field in response to increasing developments and reliance on instrumental analysis. Analytical chemistry as we know it today is the result of the major shift in nature it experienced over the second half of the 20th century. Previously, single-analyte wet chemical analysis procedures relied on hot-plate sample preparation. These procedures were gradually replaced by sophisticated instrumental methods that required equally


3

sophisticated sample preparation [3,4]. A scientist from the 1950s would be amazed at the progress made in analytical instrumentation but would feel quite at home with many of the sample preparation techniques in contemporary use [5]. Efforts at introducing novel sample preparation concepts have frequently been ignored by practitioners, who were explicitly required by law to use the approved traditional methods. Problems associated with these traditional sample preparation methods such as those arising from the use of toxic organic solvents and of multi-step procedures that often result in the loss of analytes during the process, frequently make sample preparation the greatest source of error in an analysis and preclude integration with the rest of the analytical process. This picture is about to change for many reasons, not all of which are driven by science. Thus, legislation will restrict or ban the use of several common solvents such as chlorinated hydrocarbons; also, the costs associated with the use, transport and disposal of solvents in general these days provide an economic incentive to minimize solvent consumption. Renewed awareness of the pollution and hazards caused by hydrocarbons, and carcinogenic effects, have promoted international initiatives to eliminate the production and use of organic solvents on which many current sample preparation methods rely. This phasing out of solvents is poised to induce a major change in analytical methodology and also provides an opportunity to formulate practical alternatives to existing sample preparation methods. Recycling pure solvents for trace analysis causes more problems than it solves and is generally inviable recycling analytical solvents for re-use in less demanding areas of chemistry such as organic synthesis could be viable if integrated solvent management programmes were introduced. The current fashion, in any case, is the development of new, or the improvement of old, sample preparation methods that reduce solvent consumption significantly. The attention given to sample preparation technology has brought it into the limelight much more so than in the past, such that it has become an acceptable area of academic interest but rarely of funding. Users currently know much more about traditional sample preparation procedures than they did not long ago; also, they are developing an increasing awareness of the influence of sample matrix characteristics on, for example, analyte extractability in terms of a fundamental understanding of extraction kinetics and thermodynamic analyte-matrix interactions. The greatest hurdle that keeps practitioners from adopting new analytical methods is the expense, in terms of both capital cost and training requirements, involved. An ideal sample preparation technique should be solvent-free (or, at least, use as little solvent as possible), straightforward, inexpensive, efficient, selective, easily automated and compatible with a wide range of m a t r i c e s - and applications as a result; it should allow simultaneous separation and concentration of the target components, and be amenable to on-site use. New sample preparation technologies have been slow on the uptake despite their proven advantages over some of the older technologies. In spite of many strong driving forces such as increased sample loads, decreasing skilled labour force, worker safety and less exposure to chemical hazards, the need for enhanced productivity, better quality data with increasing regulatory constraints, and the greater need for information management, automation of sample pretreatment and integration of information management into the analytical process have been accepted with reluctance. Notwithstanding all these adverse

4


impacts, new sample preparation techniques are on the horizon and are being introduced with strength. Automation of sample preparation is a clear present trend meeting the needs of current analytical chemistry and implicitly included among the research priorities of the 21st century [6]. Automation was recognized as the buzzword in ACHEMA 2000 (the 26th Exhibition Congress, International Meeting on Chemical Engineering, Environmental Protection and Biotechnology, held in Frankfurt, Germany, from May 22-27, 2000). This fashion has reached both the process industry and the laboratory. Connection to the lnternet or an Intranet was recognized as essential during the meeting. The new developments in laboratory devices placed special emphasis on cost-cutting, low maintenance and automation. This also applied to sample preparation having a major influence on the quality of the results. Even in small laboratories, attempts are being made at automating the often laborious sample preparation process. Flexible structure and easy-to-use software are indispensable if an analyser is to become established in practice. As an ongoing experiment, the SamplePrep Web, a central resource site on the Internet, was created at Duquesne University in the past decade with a view to taking advantage of the immediacy and global nature of the information superhighway. The resources held on the SamplePrep Web and other, related Internet sites, were intended to provide support for sample preparation in aspects such as sample dissolution, sample extraction or leaching, reaction chemistry, applications, synthesis, literature, standards and clean chemistry. Eventually, the site would be linked to an electronic journal on sample preparation that would help focus effort on this important area. Such support was planned to be an on-line electronic library responding to the perceived need for rapid communication of ever-changing ideas about sample preparation. Clearly, sample preparation technologies would hopefully mature more rapidly and with more stability if support was provided early, and new, global information resources could be the perfect vehicles for dissemination. To this end, scientists were invited to contribute and participate in order to help in the rapid development of this area. Unfortunately, this call had little response and the SamplePrep Web has not been revisited since 1998.

1.3. PROBLEMS ENCOUNTERED IN AUTOMATING SAMPLE PREPARATION Despite the progress, sample preparation continues to be the bottleneck of the analytical process as, only in the past 10-15 years, has its automation been actively a d d r e s s e d - by contrast, advances in automated chromatographic and spectroscopic equipment started one or two decades before. The reasons why SP remains the bottleneck of the analytical process vary among experts [7], but the most widely endorsed are as follows: (a) As a rule, SP has always been viewed as a manual technology, often regarded as "low-tech", and usually assigned to the least trained staff. (b) The addition of instrumentation to a traditionally non-instrumental step requires chemists' conversion to new techniques and chemists are somewhat reluctant to quickly accept new technologies.


5

(c) Sample preparation has little academic appeal and is rarely recognized as an analyt(d)

(e)

(f)

(g)

(h)

(i)

ical chemical area by academe. As a result, many universities have shied away from investigating it. In industry, the chemist's role is thought to be more "high-tech": to support the use of the analytical instruments as well as analyte detection. Industrial chemists spend most of their time evaluating and implementing new analytical technologies and generating and interpreting data. In addition, analytical techniques are more generic in nature; by contrast, SP can be done in many ways, a number of which are samplespecific. There are not many new concepts for the automation of SP, and early attempts tended to mimic manual operations. These approaches were unsuccessful and expensive as many of the manual procedures were not amenable to automation. Many of the major instrument manufacturers do not identify SP automation as important and it is mainly the smaller firms that have become niche players and provided the required instrumentation. For this reason, most of the early instruments were stand-alone SP modules that could not be integrated with analytical equipment as parts of the analytical process. The large firms, however, are slowly beginning to introduce automated equipment for SP. Robotics has been a revolutionary development in the context of SP automation; the implementation of robotic technology, however, is quite difficult and poses strong technological demands. Clearly, robotics and laboratory unit operations constitute the first insight into flexible automation of SP. The acceptance time of new technologies is a major constraint in SP. Widespread acceptance of a new technology usually requires a long time (often 10 years or more). Also, the time needed to validate the ensuing methods further restricts acceptance of the new technology. Regulatory agencies (particularly in the USA) are very slow to adopt new technologies, mainly as a result of most regulated methods being prescription- rather than performance-based. The inertia of most workers, usually reluctant to change and new training, is one other reason for slow acceptance. Financial payback is another major factor in the acceptance of the newer technologies, which are usually expensive. Whether the purchase of laboratory automation equipment is justified is largely dependent on the specific intended application and is actually a combination of both financial and strategic benefits.

All these constraints have restricted the development of automated sample preparation devices.

1.4. BATCH VERSUS SERIAL APPROACHES TO AUTOMATED SAMPLE PREPARATION Automated sample preparation can be accomplished by using a batch or a serial approach. In the batch mode, multiple samples are prepared and then transferred to the analytical instrument for measurement. In the serial mode, samples are prepared one at a time and the SP device is connected (integrated) with the analytical instrument only.

6


From a temporal viewpoint, it is assumed that, when the SP time is equivalent to or less than the analysis time, then a serial method is preferable. Serial methods are also more useful with samples where the assays are time-dependent or when there is a stability question of the prepared analyte (e.g. with labile samples or matrices). Examples of better application of serial techniques are OPA-amino acid derivatives for high pressure liquid chromatography (HPLC) analysis, where some derivatives have very short half-lives, and for the determination of moisture, where batching of assays could expose the samples to moisture in the environment and give rise to spurious results. When the sampling cycle time is very long, then serial SP makes more sense. Two extreme examples are environmental early warning analysis of contaminated water streams, where samples are collected every 4-6 h, and fast sample preparation procedures such as microwave digestion. In the latter case, collecting the sample directly from the microwave vessel on-line makes a great deal of sense some microwave digesters, however, are integrated with the analytical instrument to perform that task. From the viewpoint of sample availability, if limited sample is available, the user may feel more confident in preparing one sample at a time; by contrast, batch preparation is to be preferred for abundant, complex samples. Regarding the equipment subsequently used, off-line SP makes sense when the instrument is very expensive (e.g. a GC-MS, which one would not want to "tie up" during routine use). In general, the serial mode will require the development of low-cost analysers. Where this mode has its greatest potential is in feedback control led systems, which enable real-time (or pseudo real-time) process monitoring. Batch systems are more easily implemented in laboratories with routine loads and less so in laboratories that require a high flexibility or mixing and matching SP and instruments. One other situation where the batch mode might make sense is when a wide variety of SP techniques feed a wide range of analytical instruments. The type of SP procedure involved dictates whether the batch or serial approach is required. For example, centrifugation and microwave-assisted digestion tend to require batch processes. On the other hand, new technologies such as those of axial centrifuges and the new flow-through microwave digesters have revealed that even traditionally batch steps can be made serial. The batch environment is often an accommodation of the human operator in the laboratory rather than the most appropriate way for a given chemical-instrumental system. Frequently, batch methods are not science-based but developed by the constraint of an eight-hour working day, so phrases such as "let stand overnight to settle" or "allow to digest for 1 h" are common in these procedures. Automated systems are not so strongly constrained and should not be penalized with human limitations. The human element also dictates the use of the batch mode because of our limited ability to keep track of too many events. In short, automated sample preparation is changing most SP protocols and giving a new face to this step of the analytical process, which was formerly envisaged as a clumsy, unproductive step.


7

1.5. DEFINITIONS AND R E S T R I C T I O N S OF T E R M MEANINGS This section discusses some terms that are very common in the context of solid sample treatment but are not always properly used or understood.

1.5.1. Sample preparation and sample pretreatment "Sample preparation" is a term in wide use at present. There is, however, some confusion with "sample pretreatment" as no clear-cut boundary between the two exists, nor has where sample preparation ends or what precedes sample treatment been clearly established. To the authors' minds, sample preparation includes rendering the sample or, rather, the target analytes contained in the original s a m p l e - ready for insertion into the measuring instrument and can involve one or more steps such as dissolution or leaching (with solid samples), clean-up, preconcentration, derivatization, etc. Sampling is seemingly excluded from sample preparation as this designation assumes that the sample already exists as such before preparation starts. On the other hand, sample pretreatment can be envisaged as the first step of sample preparation or as a step preceding one other specific action (e.g. a pretreatment for subsequent liquid-liquid extraction or insertion of a solution into a chromatograph), which, in the case of solid samples, can be sample dissolution or leaching in a more or less selective manner of the target analytes. It is unclear when such a step finishes and when detection starts in the vaporizer or atomizer in electrothermal treatments, and so is in glow-discharge and laser-induced breakdown methods. When one of these approaches is coupled to, for example, ICP-MS, is the ionization step in the plasma also a part of the pretreatment or should it be considered a part of the detection step? In many cases, the boundaries between steps are blurred and the names such steps receive within the framework of the overall analytical process are confusing. In dealing with the subject matter of this book, solid samples, the term "sample pretreatment" is used to refer to the step whereby the analytes are isolated from the whole matrix and placed in usually a solution.

1.5.2. Solid sampling One other terminological controversy arises from the ordinary usage of "solid sampling" in relation to solid samples delivered to an electrothermal device, acting as electrodes in glow-discharge applications or being "shot" by a laser. Is it correct to use this designation after stating that "the sample is weighed and placed in the graphite tube or L'vov platform", "the sample is placed in the glow-discharge chamber" or "the sample is positioned on the XYZ translation stage for subjection to laser shots"? When does sampling take place in these techniques, before or after the sample is introduced into the specific device used? Because no universal consensus exists as regards the use of "solid sampling" to designate the particular step involved in each case, this book uses it consistently throughout.

8


1.5.3. Digestion versus leaching

The word digestion has several, rather different meanings. Thus, the Oxford English Dictionary (OED) defines it as "the operation of dissolving a substance by the action of heat and moisture" [8], whereas Webster's Third New International Dictionary (WTNID) provides the definitions "the process by which a material is softened or decomposed by heat or moisture or a chemical, often under pressure" and "the process by which soluble ingredients are extracted from plant or animal materials by warming a liquid" [9]. The last definition is fairly close to that for leaching in both WTNID ("the process or an instance of separating the soluble components from some material by percolation") [9] and the OED ("subjecting to the action of percolating water with a view to removing the soluble constituents") [8]. In order to set a boundary between both terms and adhere to the most frequent usage patterns in the literature, "digestion" is used in this book when all the components of the solid sample are dissolved under drastic conditions (viz. with the aid of heating and an acid or base) and "leaching" when only partial, selective dissolution is sought, using milder conditions. 1.5.4. Use of a leaching step versus a digestion step

Based on the most widely accepted meanings of leaching and digestion, the former is always desirable when possible. Partial dissolution of the sample with complete removal of the target analytes provides a less complex liquid and the possibility to avoid interferences. It should be noted, however, that complete dissolution of the analytes must be ensured if the target analytes are to be quantified with adequate precision. Special care must be exercised in applying a method developed with spiked matrices to natural samples rather than with real samples as opposed to prepared or artificial samples since the opposite of "real samples" would be "imaginary samples". Complete dissolution, which does not always require digestion, is usually easier to accomplish than leaching; however, the increased complexity of the latter is usually offset by the fact that it provides analyte solutions which are easier to process. 1.5.5. Leaching versus extraction

One other controversial term in wide use is extraction, which is defined as "the act to separate or otherwise obtain (as constituent elements or juices) from a substance by treating it with a solvent, distilling, evaporating, subjecting to pressure or centrifugal force, or by some other chemical or mechanical process" by WTNID [9] and as "the action to obtain (elements, juices, etc.) from a thing or substance by any chemical or mechanical operation" by the OED [8]. Extraction is thus used as a synonym for, and virtually always in place of, leaching. Thus, it is rather unusual to find the designation "supercritical fluid leaching" even though "supercritical fluid extraction" is much more general and less precise. In this book, "extraction" is used mainly to refer to steps in which a solid sample is partially dissolved to remove target species, but just because this is the more common term of the two, in fact, the authors greatly favour the use of


9

"leaching" as it is more accurate (it reflects the fact that it is a solid sample that is treated to dissolve a portion thereof). The term "lixiviation" is synonymous with "leaching".

1.6. SOME NOTES ON THE B O O K C O N T E N T S

Clearly, the variety of solid samples that can be subjected to some pretreatment precludes the development of a universal device for processing any type of sample whatever its nature. Substantial efforts have been made, however, at adapting commercially available equipment to as many types of samples as possible (liquids included) with a view to expanding their scope. Two cases in point are supercritical fluid extraction with CO 2 in the presence of a modifier for removal of polar, even ionic, compounds; and radiofrequency-assisted glow-discharge sampling, which enables sputtering of nonconducting materials without the need to "deceive" the technique by mixing the sample with a conducting support. The primary aim of this book is to provide readers interested in solid sample pretreatment with an overview of available techniques for development of this step of the analytical process. The title of the book is intended to reflect that it is mainly concerned with the dissolution or removal of target analytes from solid samples. Once they have selected the technique most closely fitting their intended purpose, readers can obtain a deeper knowledge about the technique of choice in the specialized literature in fact, providing a thorough description of each of the wide variety of sample pretreatment techniques available at present was obviously outside the scope of a book like this. In fact, only those aspects that can be illustrated with reasonable concision are dealt with specifically in it. For identical reasons, the book does not touch on the subsequent steps of the analytical process. The authors therefore assume that the reader will be acquainted with the general principles of chromatography in its different variants, as well as with those of commonplace molecular optical and electroanalytical techniques, and atomic and mass spectrometries. Although no explicit separation has been made, the devices and techniques dealt with in this book have been grouped in two different, broad categories, namely: (a) Those that allow samples to be pretreated in such a way that the target analytes are released from the matrix (e.g. those based on ultrasounds, microwaves, superheated liquids and supercritical fluids, which are discussed in Chapters 3 to 7) or help release the analytes for a subsequent treatment such as dissolution or leaching prior to instrumental measurement (e.g. freeze-drying, which is the subject matter of Chapter 2). (b) Those using electrothermal vaporizers or atomizers, a glow-discharge, laser ablation or laser-induced breakdown (with which Chapters 8 and 9 are concerned), where the pretreatment is integrated with detection, whether directly or via a plasma (ICP or DCP) source. Because of their special features, workstations and robotic stations are dealt with separately (in Chapter 10) from the techniques in the previous two groups.

10


Obviously, not all techniques involving solid samples are included in this book. Thus, such techniques as neutron activation analysis (INNA) and X-ray photoelectron spectroscopy (XPS), which require equipment inaccessible to many laboratories, are only considered in comparisons with other techniques based on more affordable instrumentation but yielding similar results. Some readers may argue that, for example, laser ablation or laser-induced breakdown spectroscopy are uncommon in analytical laboratories at present; however, the authors have decided to cover them on the grounds of their high potential. Also, the short chapter devoted to freeze-drying is intended to draw the readers' attention to a technique that is rarely regarded as a form of sample pretreatment but deserves special recognition on account of its extensive use. The authors have avoided the inclusion of exhaustive application tables, which would have unduly lengthened the book. Rather, only those references considered to be essential to illustrate some theoretical or practical aspect of the different techniques, and others representative of the state of the art in each, have been included. For comprehensive tables compiling applications or some other aspect, interested readers are referred to the reviews periodically published in specialist journals. Although sample preparation may sound archaic and alchemy-related in a time where the buzzwords are terms such as genomic or proteomics, both updated and emergent sciences and technologies continue to rely on analyses and, ultimately, on sample pretreatment.

REFERENCES

1 2 3 4 5 6 7

P. Quevauviller, Trends Anal. Chem., 17 (1998) 289. J. O'Donnell, Internat. Lab. News, December (1999) 10. B. Rosenkranz, P. Quevauviller, J. Bettner, Internat. Lab., May (2000) 20. S. Slaets, E Adams, Anal. Chim. Acta, 414 (2000) 141. J.L. G6mez-Ariza, D. Sanchez-Rodas, I. Giraldez, E. Morales, Talanta, 51 (2000) 257. Technical Report, Environ. Sci. Technol., 31 (1997) 20 A. R.E. Majors, LC-GC, 13 (1995) 742. 8 Oxfi~rd English Dictiona~, 2nd edn, Oxford University Press, Oxford (1989). 9 Webster ~ Third New International Dictiona~, Merriam-Webster, Inc., Chicago, IL ( 1981).

Chapter 2

Analytical freeze-drying 2.1. INTRODUCTION Freeze-drying, called "lyophilization" by the pharmaceutical industry, is a drying technique by which a product is solifidied by freezing and the solvent that contains it (usually water) is evaporated by sublimation (a chemical phenomenon) upon heating. The transformation of the solid phase into the gas phase takes place with neither the water nor the solvent passing through the liquid state. During the process, the product loses more than 90% of the water it initially contains. One feature of freeze-drying that singles it out from other dehydration techniques is that dehydration takes place from a frozen - - and hence less chemically active - - product and in vacuum which minimizes the effects of oxidation. These assets have made freeze-drying a widely accepted technique for processing heat-labile products such as enzymes and proteins for long-term storage. One other salient feature of freeze-drying is that the product retains its original texture and regains its initial morphology upon addition of an appropriate amount of a solvent (usually water). In fact, freeze-drying avoids the denaturation caused by the heating procedures used in conventional drying methods. The advantages of freeze-drying have been exploited since ancient times. Thus, the Peruvian Incas of the Andes increased the stability of their food reserves by storing them at the summits of Machu Picchu. There, the low prevailing temperature froze foods and water, in the form of ice, gradually evaporated by virtue of the low atmospheric pressure. However, the process was not made commercially available until World War II, where it gained widespread use as a means of preserving the blood plasma needed at front lines. Shortly afterwards, the process was applied to penicillin and freeze-drying was recognized as a major scientific technique for preserving biological samples. Freeze-drying has a broad scope of application, particularly in the food and pharmaceutical industries. Freezing immobilizes the sample, thus allowing it to retain its original form; also, the absence of water prevents microbial growth and chemical changes leading to deterioration of the sample. Because water sublimates so readily, the conditions needed to freeze-dry a food do not remove most other constituents (e.g. acetaldehyde, which gives citrus fruits part of their flavour). There are, however, many other possible uses for freeze-drying that include the preservation of microbial cultures, the restoration of books and other objects damaged by water, the concentration and recovery of reaction products, and the preservation of whole animal specimens as a form of taxidermy.

12

Analytical freeze-d~ing

The freeze-drying technique is also used in the analytical laboratory. Two additional benefits make it attractive to analysts, namely: (a) because the removal of water allows samples to be subsequently reconstituted in a minimal volume of liquid, a preconcentration effect is achieved; and (b), even more important, because of their texture, freeze-dried materials are very easily attacked by solvents. The process can be optimized for a specific purpose by using an appropriate temperature, pH, reagent addition sequence and time. One major disadvantage of freeze-drying, however, is that some volatile compounds may be removed from the sample matrix during the process. The conditions leading to the freeze-drying state can only be established with special equipment. Such equipment is currently available in a wide variety of models and sizes that range from laboratory-scale apparatus to industrial production systems. This chapter deals with the different aspects of the freeze-drying process, beginning with the steps involved and the conditions influencing them. The state of the art in freezedrying technology and equipment is then reviewed, and the aims and major analytical chemical applications of the technique (particularly as regards the pretreatment of solid samples) are discussed.

2.2. STEPS OF THE FREEZE-DRYING PROCESS

The freeze-drying process consists essentially of three steps, namely: (a) Freezing, by which most of the water contained in the sample is converted into ice by cooling. A small fraction of the water, however, is tightly bound to the sample matrix and is not frozen, the amount of water that remains in the liquid state depends on the initial composition of the sample and on the temperature used. (b) Sublimation, by which the ice formed in the previous step is converted into vapour by input of energy. The passage of solid to gas takes place with no intermediate conversion of water to the liquid state. (c) Desorption, by which the water tightly bound to the matrix (viz. sorbed water) is removed from it when the water pressure of the solid-associated water equals the water pressure of the condenser. 2.2.1. The freezing step

The first step in freeze-drying a product involves converting it to the frozen state. Any alterations of the sample during this step should be avoided. Freezing has three major effects on the sample with strong implications on the outcome of the freeze-drying process; thus, it causes partial dehydration of the sample while preserving its initial structure and determining its morphology. During freezing, most of the water in the sample is converted into ice; as a result, the solids in the remaining liquid become more c o n c e n t r a t e d - or, conversely, the solids are partially dehydrated. The ice thus formed remains in the sample, so freezing only causes a physical separation between the water transformed into ice and that remaining in the liquid state and containing dissolved solids. The ice is effectively separated from the sample matrix during the sublimation step. If the freezing step is conducted in a stand-alone

Analytical./keeze-do:ing

13

apparatus rather than in the freeze-dryer proper, then the temperature of the sample should be maintained in transferring it from the freezing device to the dryer. The extent of solid dehydration achieved with freeze-drying depends on the particular type of material and on the temperature reached by the sample during the freezing step. In any case, some water (containing dissolved solids) will always remain unfrozen in the sample. Effectively preserving the initial structure of the frozen sample relies on a combination of stiffness in the frozen and unfrozen regions. Ice is quite resistant to deformation, so it acts as a support for the unfrozen region - - the stiffness of which is more widely variable. The amount of ice formed and that of material which remains unfrozen depend on the sample temperature and the initial solid concentration in the sample, which are thus the two variables most markedly influencing the efficiency of ice as a support. Sample morphology in this context refers to the spatial distribution of the sample components following freezing. Too rapid cooling produces a large number of crystals oriented in different directions, whereas too slow cooling yields increased amounts of elongated crystals oriented in the same direction and less restrictive channels in the matrix during the drying process. The small, disoriented ice crystals formed in viscous samples by virtue of the restricted water motion, hinder mass transfer during the sublimation step, thereby resulting in less efficient drying and hence in samples more liable to structural changes. The freezing temperature also influences crystal size and hence the rate at which sublimation occurs. It is therefore essential to establish the best freezing conditions for each product. The determination of freezing points or eutectic points is probably the most complex task in the freezing protocol. The presence of any liquid constituent other than water in a liquid matrix lowers its freezing point. It is thus very important to pre-freeze the product to below the eutectic temperature prior to freeze-drying proper. In fact, any small pockets of unfrozen material remaining in the product may expand and compromise the structural stability of the freeze-dried product. The freezing points of biochemical products containing hygroscopic molecules (e.g. sugars, protein complexes) are well below zero by effect of some water molecules being tightly bound to molecules of the dissolved product. Eutectic points are usually determined from electrical resistivity measurements. As a rule, products should be frozen below -20~ for no less than 3--4 h. In some cases, freezing or freeze-drying a highly concentrated solution may prove difficult and require diluting the sample prior to freezing so that an adequate amount of ice is formed in the freezing step. 2.2.2. The sublimation step

The sublimation step removes the water molecules that were separated from the solids as ice crystals in the freezing step. Sublimation is the process by which solid water is directly transformed into vapour water. When sublimation occurs during the freeze-drying step, it is commonly referred to as the "primary drying process". The sublimation step does not affect unfrozen water associated to the solids (sorbed water), which is removed during the desorption step.

14

Analvtical freeze-d~ing

TABLE 2.1 VARIATION OF THE WATER VAPOUR PRESSURE WITH TEMPERATURE Temperature (~

Water vapour pressure (mbar)

0 -10 -20 -30 -40 -50 -60 -70 -80

6.104 2.599 1.034 0.381 0.129 0.036 0.011 0.0025 0.0005

Sublimation always starts at an open surface and then moves inwards into the sample. After some of the ice is sublimated, the sample exhibits two distinct regions, namely: the dry layer (from which ice crystals have sublimated) and the frozen layer (where ice crystals are still present). These two regions meet at the so-called "ice interface", "sublimation interface", "freeze-drying interface" or, simply, "interface". The sublimation step requires very careful control of two of the key variables of the freeze-drying process: temperature and pressure. While sublimation can occur at atmospheric pressure, the process is rather slow because the gas molecules from the ice must find their way through the atmospheric gases that are bombarding the surface of the ice. This slow process by which the water molecules leave the ice surface is known as "diffusion". The rate of sublimation of ice from the frozen product depends on the difference in vapour pressure between the product and the ice collector. It can be increased by decreasing the pressure over the ice surface. This can be accomplished by placing the frozen material in an evacuated chamber, where molecules will migrate from the sample to an area of lower pressure. Although the extent to which the pressure is reduced increases with increasing vacuum in the chamber, there is relatively little change in sublimation rate of the water from the ice surface. Only when the pressure in the chamber becomes less than the pressure of the ice is there a marked increase in sublimation rate. The vapour pressure of the ice is dictated by the ice temperature. A comprehensive list of temperatures and vapour pressures is given in Table 2.1. It is extremely important that the temperature at which a product is freeze-dried is balanced between the level that maintains the frozen integrity of the product and that which maximizes its vapour pressure. Such a balance is a key to optimal drying. The typical phase diagram of Fig. 2.1 illustrates this point. Most products are frozen well below their eutectic or glass transition point, and then the temperature is raised to just

Analytical fi'eeze-drying

15

u~ n;

03 03 w

SUPERCRITICALFLUID

221 .

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.

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.

.

.

6.03.10 1

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LIQUID

so o/ 1

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CRITICAL POINT Pc - 221 atm r = 0.32 g/ml

_[_ _ _ / , _ _ ~ bp I I I

VAPOUR

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99 100

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TEMPERATURE, ~

Fig. 2.1. p-T diagram showing phase equilibrium lines, the triple point (tp), boiling point (bp) and critical point for water.

below this critical temperature, the products being subjected to a reduced pressure. At that point, the freeze-drying process is started. Some products such as aqueous sucrose solutions can undergo structural changes during drying that result in so-called "collapse". Although the product is frozen below its eutectic temperature, warming during the freeze-drying process can affect the structure of the frozen matrix at the boundary of the drying front. This causes the matrix structure to collapse. In order to prevent collapse of sucrose-containing products, the temperature must be maintained well below a critical collapse threshold during primary drying. For example, the collapse temperature for sucrose is - 3 2 ~ (Table 2.2 shows the temperatures for miscellaneous compounds). One problem arising from collapse of the matrix structure is that, when a diluent (e.g. water) is added to reconstitute the product, the solutes may require an extended period to return into the solution, so the product will not dissolve so rapidly as in a freezedried solution. Additional problems associated to matrix collapse arise from excessive

16

Analvtical freeze-d~ing TABLE 2.2 COLLAPSE TEMPERATURES FOR SELECTED PRODUCTS AND SUBSTANCES Substance

Collapse temperature (~

Dextran Ovalbumin Coffee extract Orange juice Inositol Lactose Maltose Sucrose Lemon juice Glucose Apple juice Sorbitol Fructose

-9 - 10 - 20 - 24 -27 -32 - 32 - 32 - 36 -40 -42 -45 -48

moisture, which may result in decreased stability, a loss of potency or even chemical reactions between solids yielding a turbid solution. Energy, supplied in the form of heat, is the third key variable in the freeze-drying process. The transformation of ice into water vapour requires energy. In fact, almost as much energy is required to sublimate one gram of water from the frozen to the gaseous state as is needed to freeze one gram of water. Sublimating one gram of ice entails using about 675 calories. All other conditions being appropriate, heat must be applied to the product to facilitate the removal of water in the form of vapour from the frozen product. The amount of heat used must be carefully controlled as applying more than evaporative cooling in the system can remove will warm the product above its eutectic or collapse temperature. Heat can be supplied in various ways (e.g. directly through a thermal conductor or from the environment).

2.2.3. The desorption step The sublimation step separates the ice crystals formed during the freezing step. When an ice crystal forms, that which remains is the concentrated solute phase called the "dry layer". This will become the freeze-dried material at the end of the process. Immediately following passage through the interface, however, the solids in the dry layer still contain a substantial amount of water (about 25-30 g per 100 g of solids), which continues to be strongly bound to the solids. Most sample materials will not be structurally or chemically stable unless most of this water (called "sorbed water") is removed. The process


A)

,zll

/3

, ,z

17

B) T1

T2

IZ

o o

ro

#. 2

u.I n,,' :) I-

or)

o v

P = A.Po

Aw = P/Po

Fig. 2.2. Typical water sorption isotherms (T~ > T, > Tl). (A) Based on water pressure. (B) Based on water activity. A,, water activity, p water pressure exerted by the sorbed water molecules in the solid at a given temperature, P0 water pressure over pure water at the same temperature. by which this more strongly associated water is removed is called "desorption" (or "secondary drying"). Sorbed, associated or bound water is water that exerts a lower pressure than pure liquid water at the same temperature. This pressure reduction results from binding of the water to the solids, the binding strength dictating to what extent the pressure is reduced. Sorption isotherms (Fig. 2.2) expose the relationship between the water pressure and water content of a material at a given temperature. The sorption isotherm for the material varies with temperature in such a way that, at each sample moisture level, the water pressure increases with increase in sample temperature. The most severe problem encountered in this step of the freeze-drying process arises from the need to supply the solid-bound water molecules with energy (heat) in order to effectively evaporate them. One way of solving the problem is by raising the pressure in the system provided the stability of the product is not compromised which can be accomplished by injecting an inert gas for some time and then re-evacuating. The inert gas provides the system with molecules that drive the energy from the heat source to the bulk product and act as brushes by transferring the vapour molecules from the product to the condenser.

2.3. VARIABLES INFLUENCING THE FREEZE-DRYING PROCESS

The two variables most strongly affecting the freeze-drying process are pressure and temperature. Temperature (or, rather, a temperature difference) is the driving force for heat transport. Energy moves from locations of higher temperature to locations of lower temperature. Similarly, pressure differences are the driving force for mass transport. The

18

Analytical freeze-drying

influence of these two variables on the different steps of the freeze-drying process, and their relationship to other influential variables, are discussed below. The freezing step is conducted at atmospheric pressure. It is crucial to ensure that the product will be thoroughly frozen by the end. This entails maintaining the temperature below the eutectic or collapse point of the product. In order to freeze the product, the shelf temperature is reduced to a level well below the collapse temperature. Depending on the dimensions of the container and on the fill volume of the product, the product temperature is maintained at shelf level for a specified period of time to ensure the formation of a completely frozen matrix. In general, the term "shelf temperature" as applied to freeze-drying refers to the shelf fluid rather than to the shelf surface temperature. The shelf temperature may represent the fluid temperature in a reservoir, the inlet or outlet temperature of the fluid to the shelves or the fluid temperature in the inlet or outlet of the shelf manifold. To avoid ambiguity, it is preferable to use "shelf temperature" to refer to a measured shelf fluid temperature. In the absence of a product temperature monitor, the formation of a completely frozen matrix relies on the length of time the product is on a shelf at a given fluid temperature. For vials loaded into the dryer on trays, additional time must be allocated to compensate for the variation in the heat transfer properties of the trays. The sublimation step begins after the freeze-dryer attains the required condenser temperature and chamber pressure. The shelf temperature is increased such that sublimation of the ice in the product matrix occurs and the product temperature [1] is significantly lower than the collapse temperature to ensure a completely frozen matrix throughout the sublimation step. Ideally, the product temperature during this second step of the process will be directly dependent on the shelf temperature and chamber pressure. Hence, in order to maintain a given product temperature, one cannot alter the chamber pressure without introducing a compensating change in shelf temperature. This relationship was demonstrated when the temperature of ice in a stainless steel tray increased from - 4 7 ~ to - 2 4 ~ as the pressure in the chamber was raised from 55 mTorr to 500 mTorr at a shelf-surface temperature of - 1 6 ~ [2]. Also, a change in the shelfsurface temperature at a given, constant pressure, produces a concomitant change in product temperature. For example, lowering the shelf-surface temperature decreases the product temperature at a fixed chamber pressure. Validation of the freeze-drying process has been found to rely upon the establishment of a range of product temperatures yielding a product with properties falling within a set of prescribed limits [3-5]. It is therefore essential to ensure that the product temperature is maintained within such limits during the sublimation step. One way to ascertain that the sublimation step has completed is by monitoring the pressure rise in the drying chamber as a result of temporarily isolating it from the pumping system [6]. An increase in chamber pressure at a given shelf pressure will result in an increase in product temperature. Such a temperature increase may exceed the collapse temperature and result in collapse or, under extreme conditions, in meltback of the product. In the absence of monitoring of the product temperature, this method is only applicable if the pressure in the chamber does not exceed the vapour pressure of ice at the collapse temperature. Because the shelf temperature during primary drying will

Analytical freeze-dr3,ing

19

exceed the collapse temperature, ensuring that the product temperature does not exceed the collapse temperature can only be accomplished by monitoring the product temperature. The pressure rise test has been found to alter the drying rate [6]. The characteristics of the dryer can also affect the product temperature if spurious pressure values are measured. Depending on the thickness of shelf-to-shelf spacing, pressure differentials may exist across the shelf. Such differentials mean that the product temperature in the middle of the shelf may be significantly higher than that on its edge near the inlet to the condenser chamber. The magnitude of the pressure differential increases with decreasing distance between shelves. In addition, the differential varies from process to process depending on the rate of sublimation of the product. Differences in sublimation rate across the shelf during the sublimation step have indeed been observed [7]: the drying rate was found to be lower in the middle and back of the shelf than in the front. These results suggest that a substantial amount of radiant energy might be transferred from the dryer door to the product. Product monitors placed at the front of the dryer would indicate product temperatures not representative of the general population of the product on the shelf. The desorption step serves to reduce the residual moisture content in the product such that it will no longer support biological growth or chemical reactions [1]. The final residual moisture content in the product will depend on its adsorption and desorption isotherms [8,9]. The equilibrium moisture content of the product depends on its temperature, the vapour pressure on its surface and its potential chemical interactions with water vapour molecules. For a given product, the final moisture content can be reduced by increasing the shelf temperature at a constant water vapour pressure or by decreasing the water vapour pressure on the product surface at a constant temperature. The completion of the desorption step is generally signalled by the product temperature approaching the shelf temperature for some specified length of time. In the absence of significant errors in chamber pressure potentially affecting the flow of water vapour from the container, pressure will not affect the product temperature as much as the shelf temperature. The main difficulty during the desorption step is that arising from changes in shelf temperature.

2.4. FREEZE-DRYING METHODS

Freeze-drying can be implemented in various ways the most common of which are manifold drying, batch drying and bulk drying. Each is used for a different purpose, the most suitable choice in each case depending on the specific initial and end products. 2.4.1. The manifold method

This method uses a turret with multiple ports (a manifold) to which flasks and vials are fitted via suitable valves (see Fig. 2.3). The product is either frozen in a freezer (by direct submersion in a low-temperature bath) or shelf-frozen, depending on the nature of the initial and end product, and also on the volume to be freeze-dried. The pre-frozen product must be immediately attached to the drying chamber or manifold to prevent

20

Analyticalfreeze-d~ing

Fig. 2.3. Manifold-based freeze-dryer. (Reproduced with permission of FTS Systems.) warming. The product container should be rapidly evacuated and the operator must rely on evaporative cooling to maintain the product temperature. This procedure is only applicable to products with high eutectic and collapse temperatures, and to small volumes at a time. Manifold drying has several advantages over batch tray drying. Because vessels are attached to the manifold separately, each vial or flask has a direct path to the collector. This eliminates some of the competition for molecular space created in a batch system and is most ideally realized in a cylindrical drying chamber where the distance between each product and the collector will be identical. In a "tee" manifold, the water molecules leaving the product in vessels farthest from the collector experience some traffic congestion as they travel past ports of other vessels. Heat input can be effected by exposing the vessels to room temperature or by using a recirculation bath. Manifold drying may be unsuitable for some products requiring strictly controlled temperatures. Vessels of variable size and closing mechanism can be attached to the same manifold in order to dry various types of product simultaneously. As the products and their volumes may differ, each vessel can be removed from the manifold separately as its drying cycle is completed. Also, the close proximity to the collector creates an environment that maximizes the drying efficiency.


21

2.4.2. The batch method The vessels used in this drying method are all of the same size, contain the same product and are placed together in a tray dryer. The product is usually frozen on the dryer tray. The product drying temperature and the amount of heat applied must be kept constant throughout the process. Usually, all the vessels in a batch are processed similarly, even though variations in the system can reasonably be expected. Thus, the amount of heat applied to different zones of the tray may vary slightly. Also, vessels located in the front portion of the shelf may be radiantly heated through the clear door. These slight variations can result in small differences in residual moisture. The batch method allows one to stopper all vessels in a lot at the same time, under identical atmospheric conditions. The vessels can be stoppered in vacuum or after backfilling with an inert gas. Stoppering all the vessels at once ensures a uniform environment in each vessel and uniform product stability during storage. Batch drying is the method most frequently used by the pharmaceutical industry to prepare large number of flasks or vials of the same product for marketing. 2.4.3. The bulk method Like batch drying, bulk drying is performed in a tray dryer. However, the product is poured into a bulk pan and dried as a single unit. Although the product is spread throughout the surface area of the shelf and may be the same thickness as products dried in vials, the lack of empty spaces within the product mass changes the rate of heat input, which is restricted primarily to that provided by contact with the shelf. Unlike manifold-based or batch drying, bulk drying allows no control of the conditions to which the product is exposed. Usually, the product is removed from the freeze-drying system prior to closure and then packaged in air-tight containers. Bulk drying is usually restricted to stable products scarcely vulnerable to oxygen and moisture.

2.5. COMPUTER-ASSISTED ANALYTICAL FREEZE-DRYING The high performance of currently available computer hardware and software has inevitably led to their application in the freeze-drying process. A carefully compiled data library can be used to determine the optimum values of such crucial variables as the pressure, temperature and time required in each step simply by inputting the composition of the sample to be processed. Below are briefly discussed the inputs one would need to enter or that should be present in the software in order to perform the three steps of the freeze-drying process as implemented by Jennings [8]. 2.5.1. Computer-assisted freezing In this first step, the computer software will require an input or a function that will compute the following minimum thermal properties of the sample: degree of

22

Analytical fi'eeze-d~ing

crystallization, collapse or eutectic temperature and degree of supercooling. This last thermal property depends not only on the composition of the sample but also on the nature of its container (a vial or tray).

2.5.2. Computer-assisted sublimation The sublimation step should be started when the matrix is in a completely frozen state. In order to ensure that the matrix will preserve its frozen state throughout the sublimation step, the computer should be provided with the collapse or eutectic temperature of the sample. Since the vast majority (99%) of samples will have a collapse rather than a eutectic temperature, the software will need to base the product temperature on some knowledge of, or means of computing that for the sample concerned. Such a frequency distribution of collapse temperatures arises from the fact that the glassy state, formed in the interstitial region of the matrix, is non-stoichiometric in composition, so the collapse temperature can vary from container to container depending on the particular frequency distribution. Based on the size (sample volume and number of vials) of the lot, the software must take into account that the calculated ideal product temperature may exceed the collapse temperature. The chamber pressure during the sublimation step (i.e. the primary drying process) has been found to be related to the product and shelf-surface temperatures [8]; however, determining the shelf temperature required is more difficult as it depends on the nature of the heat transfer fluid used to control the shelf temperature and also on the particular design of the freeze-dryer. The sample container rests on a boundary layer at the top of the shelf surface. Such a layer is a region where the flow of heat transfer fluid is minimal or zero (i.e. the fluid is stationary). As a result, the sublimation step, which involves the transfer of heat from the fluid to the shelf surface, creates a temperature gradient across the boundary layer that depends on the thermal load exerted by the sublimation process and on the nature (viscosity, thermal conductivity and flow across the shelves) of the heat transfer fluid. The temperature gradient also depends on the number of shelves, their design and build, and on the pumping capacity of the circulation pump. These variables in turn depend on the size and particular manufacturer of the freeze-dryer, so the software used should include an input of data for the materials used, and for the dryer's design and build. The computer must determine the rate at which this step develops from the heat of sublimation of the ice in the sample and the uncrystallized water present in the interstitial region. Also, it should take into account the heat transfer coefficient of the sample container - - in the case of a vial, not just a single container, but the frequency distribution of an entire lot of containers. From this statistical information, the computer can then determine the highest shelf-surface temperature where, based on knowledge of the batch size, the chances that the heat transfer coefficient of a container would result in a product temperature exceeding the collapse temperature and yielding a defective product are minimal. The frequency distribution of the heat transfer coefficients of the containers would also provide the computer with the information needed to extend the primary

Analytical fi-eeze-drying

23

drying process long enough to allow containers with coefficients falling on the lower portion of the frequency distribution to complete the sublimation step.

2.5.3. Computer-assisted desorption This step is intended to reduce residual moisture to levels allowing no microbial growth or chemical reactions of the end product. The amount of residual moisture present in a product depends on its desorption isotherms. Such isotherms in turn depend on various factors including the product temperature, pressure chamber, partial vapour pressure in the container and nature of the interaction of the water vapour with the interstitial material formed in the freezing step. The computer should be fed with information on the target sample component. For example, if the component of interest is a protein, then overdrying may alter its configuration and decrease the potency of the end product. Consequently, the computer should control not only the final product temperature but also the partial water vapour pressure and the duration of the desorption step.

2.6. LABORATORY-SCALE FREEZE-DRYERS 2.6.1. Basic components of a laboratory freeze-dryer All freeze-dryers are constructed from the same basic components, even though they may vary greatly in size and appearance. A freeze-dryer for food production of tons-per-day capacity (e.g. one for coffee) operates on essentially the same principles as a laboratory freeze-dryer used to prepare grams-per-day amounts of tissue samples for microscopic analysis. Basically, a freeze-dryer consists of a port for inserting the product, a drying chamber, a vacuum pump, a condenser, and vacuum and temperature gauges and controls. The product insertion port is usually a tray or a manifold where the frozen product is placed for freeze-drying. The drying chamber in a shelf freeze-dryer provides a thermally insulated vessel to accommodate the dryer shelves, which are specially constructed so that their temperature may be changed by passing a heat transfer fluid or gas. The gas or liquid is used to cool or heat the product. For freeze-drying in vials, the shelves can be moved so as to stopper the vials prior to opening the dryer door. The drying chamber should be equipped with a vacuum gauge in order to accurately monitor the pressure during the drying process. The freeze-dryer is equipped with a mechanical pumping system that removes noncondensable gases. With oil sealed mechanical pumps, one should be careful to operate the dryer so that no hydrocarbon vapours from the pump can backstream into the drying chamber. The condenser's main purpose is to remove the water vapour that is either sublimated or desorbed from the product. The latter is accomplished by directing the flow of vapours from the product over a cool surface. In an "internal" condenser, the condensing surfaces are located in the drying chamber; in an "external" one, the surfaces are contained in a

24


separate, insulated chamber. The principal advantages of an internal condenser are that the dryer occupies less floor space and is less expensive. On the other hand, an external condenser can be isolated from the drying chamber during backfilling of the chamber with a dry gas (e.g. nitrogen). In this way, the possibility of water vapour being transported from the condensed ice surface back to the product is avoided. The dryer should possess temperature sensors to measure the shelf and product temperatures throughout the freeze-drying process. Most freeze-dryers can be equipped with a computer system to control the pressure in the chamber and the shelf temperature as a function of time. The product temperature allows one to confirm that the shelf temperature and chamber pressure are within their preset limits.

2.6.2. Evolution of laboratory freeze-dryers The mechanical design of freeze-dryers has evolved slowly since the 1940s. Many users of freeze-drying systems realized the need for special designs; in response, they altered their laboratory equipment themselves or had the required changes made on a commercial scale. In the mid-1990s, manufacturers started to incorporate the allegedly most universally demanded variations. This introduced new control equipment and mechanical designs in freeze-dryers for research purposes. Below are comments on the most salient shortcomings of the original freezer-dryers and the ways they were circumvented.

Pressure and temperature controls" Tray-based freeze-dryers were formerly heated by means of electrical wires, the temperature being controlled by using a thermostat. The temperature of the shelves (or the product itself) was measured with thermocouples furnished with analogue indicators. Pressure measurements were read off a mercury gauge. These obsolete devices were not precise enough for control and measurement purposes or were cumbersome to use. Freeze-dryer controls have experienced the most dramatic developments in recent years. The original mercury gauge and thermostatically controlled heaters were initially replaced with analogue vacuum gauges, thermocouples and controls. Eventually, the latter two were superseded by digital devices. Digital controls were initially non-automatic. The operator was required to set the shelf temperature to be used to freeze the product (in tray-based systems) and, once such a temperature was reached, to activate the condenser cooling mechanism. When the condenser attained a temperature o f - 4 0 ~ the operator started the vacuum pump and allowed the system to stabilize; a new intervention was subsequently required to raise the shelf temperature for the sublimation step. A thermal delay in time between the temperature set by the operator and the product temperature usually existed as a result of the low pressure in the system. In order to maximize the freeze-drying efficiency, the operator had to go through many trial-and-error cycles until the amount of heat supplied was balanced with that of vapour migrating outside the unmelt product; this led to unnecessarily long freeze-drying cycles. In addition, the most widely used method for detecting

Analytical J?eeze-drving

25

the end of the sublimation step involved isolating the tray chamber from the condenser module by using a vacuum pump. A rise in chamber pressure indicated that the process was still in the primary drying step. If the valve was shut when the temperature of the product was close to its melting point, the resulting pressure drop caused the product to melt down. The thermostatically controlled heaters formerly used in tray-based freeze-dryers were gradually abandoned as they resulted in inconsistent product temperatures. Most freezers of this type currently use hollow trays through which a heat transfer liquid is passed at a controlled temperature. Operating currently available freeze-dryer models is less labour-intensive; also, modern equipment affords the implementation of some initial production stages. Once the few sample (melting point) and protocol data (final tray temperature) required is input by the operator following insertion of the product, the apparatus conducts an entire freeze-drying cycle. Pushing control buttons is all the operator has to do in order to have the system operate there is no need to continually return to the freeze-dryer during the work cycle. The fact that the dryer pressure is strictly controlled avoids a thermal gap between the tray and product temperatures. For safety reasons, the pressure is set in such a way that the temperature is 5~ below the melting point of the product. On an hourly basis, the freeze-dryer automatically uses a drain valve to allow the pressure within the system to drop. A simultaneous fall in product temperature is indicative of the presence of frozen solvent that should thus be extracted during the sublimation or primary drying step. Detecting any residual solvent in this way avoids the risk of the product melting down. Before the desorption step is started, this pressure control cycle is repeated until no further drop in product temperature is observed. The vacuum pump

The vacuum pumps initially used in freeze-dryers were of the belt-drive type and, as such, extremely bulky and noisy by current standards. At present, most research-scale freeze-dryers use direct-drive pumps, which are much quieter than the previous models and also, frequently, very small. Because they are constructed from corrosion-proof materials, they are highly flexible in operation. The low-pressure zone and the vapour trap

In the earliest commercially available freeze-dryers, the functions of the low-pressure zone and the vapour trap were both served by a chemical or ice trap. A major change in the design of the low-pressure zone was introduced in 1955. Dry ice used as coolant was replaced with mechanically cooled low-temperature condensers, which proved much easier to use and maintain. The earliest condensers were made of stainless steel. As the scope of freeze-drying expanded, a need for a chemically resistant type of trap arose. In response, titanium traps that proved much more resistant to corrosion were developed.

26

Analvtical freeze-d~ing

The closing system The devices initially used to stopper flasks on the shelves used a manually actuated screw-type mechanism that pushed the trays down. The procedure was very cumbersome and prone to introducing contaminants in the product, so it was eventually superseded by one based on air diaphragms. A bag is filled with atmospheric air to act on a plate that forces stoppers into flasks and vials. Depending on its shape, the bag can cause stoppers to seat misaligned in the vial mouths and the vials to break. If the bag bursts while being filled, the product will be contaminated. Built-in electromechanical closing devices have made air bags redundant. They avoid contamination of the product with external agents during closure and ensure simultaneous stoppering of all flasks or vials without breaking them.

Additional saJety systems Modem laboratory-scale freeze-dryers include additional devices for improved safety. Some avoid condenser overload or power outages. During freeze-drying, the condenser temperature creates a low-pressure zone in the dryer for vapour to migrate. Such a zone should therefore be maintained at a preset, low level. If such a level is exceeded, then a pressure rise in the system can cause the product to melt down. Freezer-dryers equipped with condenser overload controls automatically cool the shelves until the emergency is solved. A freeze-dryer that has gone through a power outage will continue to operate until the pressure within the system is so large that no sublimation or vapour migration will occur. Some freeze-dryers check the condenser and system pressures each time the system is reset. If both fall within the prescribed ranges, the freeze-dryer automatically continues on to the next step. However, if the conditions established during the power outage do not allow the process to continue, the freeze-dryer will automatically reffeeze the product and the condensate but will not start the evacuation system. Changes and advances in mechanical design of freeze-drying equipment and control systems have had a strong impact. Modern freeze-dryers are easier to use, require less operator intervention and are applicable to a wide variety of products. Current, automated freeze-dryers allow the initial steps of the protocol to be implemented, thereby providing the operator with more data of interest; also, they are safer and easier to operate than previous models.

2.6.3. Laboratory-designed freeze-dryers A variety of small devices has been designed to meet specific functions not effectively served by commercially available freeze-dryers. Such devices were preceded by a number of customized dryers that were developed for various analytical purposes. Thus, Nakaguchi et al. [10] designed a new drying apparatus equipped with a device for trapping evaporated substances that was used to condition biological samples prior to determining trace elements.

A nalytical freeze-drying

27

Auxiliary devices for freeze-dryers have been designed for various purposes. Thus, a tray apparatus containing several wells was developed to freeze-dry biological substances (particularly anticoagulants used in blood gas analyses). This apparatus is suitable for producing a "pledget" or single-unit dosage of predetermined values of freeze-dried biological material that can be stored for extended periods in a sterile syringe to be used as blood anticoagulant. The tray has an upper plate laminate (covered by an insulator during freezing) with rows of wells for holding a biological solution of a preset concentration and a lower laminate in the form of a sheet of conducting material for establishing a temperature gradient across the height of the tray to assist in freezing the tray contents as the stackable trays sit on the cooling or heating shelves of the freeze-dryer [11]. An instrument performing measurements of D2 (viz. the ratio of the resistivity of ice to that of a product at a given temperature) and implementing DTA (differential thermal analysis) was developed to control freeze-drying processes; the principal components are a test chamber, a cooling and heating unit, a digital computer system and a printer. It was used to evaluate the thermal features of a 20% sucrose solution [12]. Rigid and semi-rigid disposable reaction containers for measuring absorbance changes have been reported [13]. A windowed reaction chamber was adapted to transmit light and connected to an auxiliary chamber via a small opening that allowed the reagent to be forced through to trigger a reaction. The container may be used in automated assays. Also, components of a freeze-dried reagent can be stored in compartments for later analysis.

2.6.4. Commercially available freeze-drying equipment A wide range of apparatus exists that meets most laboratory needs as regards freezedrying. Below are described some of the more widely used designs.

ManiJbld-based designs These are best exemplified by the Cryodos-50 and Cryodos-80 models from Telstar (Fig. 2.4), which consist essentially of the following elements: (a) A vacuum pump furnished with a gas ballast and a cut-off valve. The valve isolates the body of the freeze-dryer from the pump when the latter is stopped and effectively maintains a high vacuum in the dryer while atmospheric pressure is restored within the pump, thereby avoiding oil backstreaming. The pump also contains an eject filter. (b) A cooling system that lowers the condenser temperature to the working level. The condensation surface is sized in such a way as to ensure condensation of the water vapour released by the product during the freeze-drying process and prevent it from reaching the vacuum pump. (c) A stainless steel cylinder located at the top of the system and equipped with eight stopcocked ports each of which is furnished with a three-way valve that allows each flask to be isolated and atmospheric pressure restored within it on an individual basis

28


Fig. 2.5. Three-way valve for isolating each individual flask and restoring atmospheric pressure with it. (Reproduced with permission of Telstar.)

Analyticalfreeze-drying

29

(Fig. 2.5). The valves allow ground-mouthed flasks to be fitted via a conical adaptor. By removing the adaptor, tubes and vials can be directly fitted to the valve. (d) The controlpanel, which includes an alphanumeric LCD display that shows the menu for the freeze-drying cycle (and the condenser temperature and vacuum pressure throughout the process). (e) Ancillary devices that allow small volumes of product contained in various packages, vials, ampoules (or even bulk products) to be processed.

Tray-based designs This type of design is aptly exemplified by the Telstar models Lioalfa 6-50 and 6-80 (Fig. 2.6). Both are compact, benchtop apparatuses that accommodate the vacuum pump and cooling system. Also, both use the same type of vacuum pump as the Cryodos models.

Fig. 2.6. Tray-based freeze-dryer (Lioalfa model). (Reproduced with permission of Telstar.)

30

Analvtical freeze-drving

The cooling system consists of one or two air-tight, one-step compressors and uses air as coolant. Like the previous ones, the Lioalfa models can be equipped with ancillary devices for processing bulk products and samples in flasks, vials or ampoules.

Miscellaneous designs Depending on the number of containers to be used at once, some freezer-dryers use a multiport turret (a manifold) or a tray module. The former is suitable for containers of varying shape or size that need not be processed simultaneously; the latter is to be preferred when a large amount of product or number of containers is to be processed in a simultaneous manner. The tray module can be equipped with an electromechanical device to close the containers after the products are freeze-dried in order to avoid external contamination. One example of this type of freeze-dryer is the model from FTS Systems. Some multi-purpose models such as Telstar's Liogamma can be used as research plants with a view to studying or storing sensitive biotechnological products or as pilot plants for the accurate reproduction of entire freeze-drying processes or the examination of freeze-drying curves of industrial use. They allow small production lots to be freezedried with a view to assessing the marketing potential of new products.

2.7. G E N E R A L AIMS OF ANALYTICAL FREEZE-DRYING A product can be freeze-dried for various purposes depending on the particular features the resulting material is required to possess. Below are discussed some of the more salient uses of the freeze-drying technique.

2.7.1. Increasing long-term storage stability The very essence of the freeze-drying process is that it is a stabilizing process. In foods and biological samples such as enzymes, blood fractions, urine, etc., freeze-drying can effectively avoid unwanted chemical or biochemical reactions and hence deterioration of the material. It is especially i m p o r t a n t - particularly with biological samples that any inherent biological activity be preserved throughout the process and recovered upon rehydrating the material. The stability of freeze-dried materials is dictated by various factors two of the most important of which are moisture and oxygen. All freeze-dried products contain a small amount of water called "residual moisture". The exact amount that remains in the material depends on the nature of the product and on the length of the secondary drying step. Residual moisture can be measured by chemical, chromatographic, manometric or gravimetric means. It is expressed as a weight percentage of the total weight of dried product. Residual moisture values typically range from < 1% to 3%. By nature, freeze-dried products are hygroscopic, so exposure to moisture during storage can destabilize them. Packaging used for freeze-dried materials must be impermeable

Analytical fi'eeze-d~ing

31

to atmospheric moisture. Storing products in low-humidity environments can reduce the risk of degradation by exposure to moisture. Oxygen is detrimental to the stability of most freeze-dried materials, so the packaging used should also be impermeable to air. The detrimental effects of both oxygen and moisture are temperature-dependent. The higher the storage temperature is, the faster a product degrades. Most freeze-dried products can be stored at refrigerator temperatures (i.e. 4-8~ Storing freeze-dried products at lower temperatures extends their shelf life, however. The shelf life of a freeze-dried product can be predicted by measuring its rate of degradation at a high temperature. This is called "accelerated storage". By choosing the proper time and temperature relationships at high temperatures, the rate of product degradation at lower temperatures can be predicted. Freeze-drying slows down the kinetic clock (viz. the rate at which a substance undergoes change). One example of a substance undergoing a slow change is rusting iron; one of a substance experiencing a rapid change is burning gun powder. While freeze-drying is not used to prevent rusting of iron or burning of gun powder, it is used by the health care industry to slow down the kinetic clock of pharmaceuticals. For example, a pharmaceutical formulation will retain at least 90% of its original potency for two weeks when stored at 4-8~ in a refrigerator. After two weeks, the potency is found to be less than 90% and the product must be discarded. This means that the pharmaceutical product would have to be prepared, bottled, shipped while being maintained at 4 - 8 ~ and administered by physician to patient all in less than two weeks. If the manufacturer shipped too much product then it would experience a financial loss as a result of the unused product being discarded. Freeze-drying converts one second at refrigeration temperature into one minute at room temperature. By changing the rate of the kinetic clock, the formulation that was only stable for 2 weeks at 4-8~ is now stable for 120 weeks (2.3 years). In slowing down the clock, the manufacturer now has time to test the product before releasing it to the public. In addition, the product no longer requires refrigeration at 4-8~ which makes worldwide distribution much easier and safer. Also, there is less chance that the product will not be available for a patient or that it will expire and need to be discarded.

2.7.2. Preparing tissues for microscopic examination Properly conditioning tissue samples for examination under a microscope usually entails their prior drying. This can be done by freeze-drying, provided the tissue preserves its structure throughout the process.

2.7.3. Preparing samples as an intermediate step in (bio)chemical analytical procedures Some types of chemical analysis require that the sample be in dry powder form, either because of restrictions imposed by the analytical system to be used or in order to concentrate the sample and boost the sensitivity of the analytical method applied. The necessary condition for freeze-drying to be applicable with this purpose is that it should not alter the sample composition, which will depend exclusively on the particular freeze-drying conditions used.

32


LONG-TERM STORAGE STABILITY

RECOVERY OF PRODUCTS FROM A DILUTED SYSTEM

SAMPLE PREPARATION AS A STEP IN ANALYTICAL PROCEDURES

PREPARATION OF TISSUE SAMPLES SUITABLE FOR STRUCTURAL STUDIES

Fig. 2.7. Selected materials typically subjected to freeze-drying and aim of the process in each case.

2.7.4. Recovering products from a reaction mixture Freeze-drying is a convenient, advantageous alternative to recovering reaction products from dilute media provided it does not alter the chemical or biological activity of the product of interest. Figure 2.7 shows selected products typically subjected to freezedrying and the main purpose of using this drying technique in each case.

2.8. ANALYTICAL USES OF F R E E Z E - D R Y I N G Freeze-drying has rarely been considered an effective choice with a view to automating sample pretreatment, possibly because of the contradictory results obtained so far. However, there is solid evidence that this technique is suitable for many purposes.

Analvtical freeze-drying

33

The analytical uses of freeze-drying can be classified into three broad categories according to target: reagents, standards or samples. This section describes the principal applications of analytical freeze-drying, particularly in relation to solid samples; some mention of liquid samples is also made as serum and urine are among those most frequently used to develop analytical standards.

2.8.1. Reagents Storing reagents poses special problems, particularly in clinical analysis. One major difficulty encountered in evaluating the results of immunochemical tests is the absence of affordable reagent standards [14]. This shortcoming has been partly circumvented by using freeze-dried reagents. The large number of patents granted to applications in this field in the 1980s testifies to the high potential of the freeze-drying technique for immunochemical analysis. Freeze-drying a reagent will only be justified if (a) the process improves its stability and (b) the reagent can be readily reconstituted. In some cases, the stability of the final, freeze-dried reagent can be improved by using a suitable additive. Most immunoassay reagent standards possess improved stability upon freeze-drying. Thus, freeze-dried IgG aggregates stored at 4~ for 4 months exhibit minimal changes in biological reactivity and physical integrity with respect to identical preparations frozen at -20~ [15]. This allows IgG aggregates to be used as reagent standards for the comparison and standardization of complex immunoassays. Freeze-dried reagents can be reconstituted by rapidly injecting the diluting solution onto the vial walls or by slowly or rapidly injecting it onto the surface of the freezedried material. The properties of the resulting reagent solution will depend on the particular method used. Thus, rhGH preparations reconstituted using the previous three methods exhibit a varying degree of gelling [16]. The method of choice in each case will be that providing the best results with the reagent concerned. In many cases, obtaining a stable freeze-dried reagent requires using an additive. For example, the horseradish peroxidase-conjugated monoclonal antibodies (mAb-HRP) used in diagnostic serology are severely altered by freeze-drying; however, supplying the sample with 0.3 M trehalose or a mixture containing 0.8% lactalbumin and 3.2% sucrose substantially increases the long-term stability of the dried product with minimal loss of biological activity upon reconstitution [17].

2.8.2. Standards Freeze-drying for preparation and storage of standards has found especially widespread application in the clinical field, particularly in relation to material standards containing a large variety of analytes. The sera used as control samples and standards in clinical chemistry should remain stable over relatively long periods of time if they are to be useful and provide reliable results. Freeze-dried serum provides no advantage over serum frozen at -30~ in the determination of species such as glucose, urea, nitrogen, sodium, potassium, total protein and

34

Analyticalfreeze-d~ing

serum glutamic oxalacetic transaminase [18]. Although freeze-drying is widely assumed to alter the protein matrix of serum through changes in the ternary structure of proteins, some results appear to contradict this widespread belief. Thus, isotachophoretic bands suggest that, while some changes in ternary structure result from freeze-drying, treatments such as dialysis and the addition of chemicals or tissue extracts during the manufacturing process can also substantially alter the protein matrix. The storage conditions, the presence of proteolytic bacteria and "aging" of serum can also induce changes in protein constituents [19]. The preparation of control serum containing lipo- and apolipoproteins is specially difficult. Thus, lipoproteins in plasma cannot be stored in a liquid state for long. Also, some lipoproteins become insoluble following freeze-drying; the resulting serum is turbid and provides spurious spectrophotometric measurements. Consequently, lipoproteins are usually removed from quality control materials in order to substantially reduce their cholesterol and triglyceride contents. Most lipoproteins are affected by freeze-drying, so they cannot be related to the original lipoproteins; this hinders their determination with electrophoretic and immunoassay methods. The denaturation of serum lipoproteins during freeze-drying can be prevented in various ways. One simple manner is by supplying the serum or control plasma with sucrose prior to freeze-drying [20]. The mechanism by which sucrose protects lipoproteins from denaturation during freeze-drying remains obscure. Possibly, the sugar displaces water from the hydration sphere during the process. The material will easily accept water during the reconstitution step, thereby facilitating reconstruction of the hydration sphere. The methods most strongly affected by the physico-chemical conditions of the target analyte are the electrophoretic determination of lipoproteins and the nephelometric determination of apolipoprotein B. With both methods, the results for the control serum cannot be distinguished from those of fresh whole serum. The ability to prepare freezedried human serum containing stepped levels of apolipoproteins A~ and B for use as reference material is of great interest on account of the prominent roles of these substances as indicators of heart disease risk [20,21]. One way of increasing the thermal and longterm stability of these lipoproteins is by adding ethyl alcohol and acetic/acetate buffer to precipitate fractions enriched with them that will withstand both the passage of time (up to 3 years) and high temperatures (up to 56~ [22]. This technology can thus be used as a preparative method for reference materials of apolipoproteins at concentrations spanning the broad ranges required by clinical applications. Apolipoprotein E, which has a direct influence on the development of cardiovascular and neurodegenerative diseases, exhibits no change in immunoreactivity upon freezedrying provided a bicarbonate buffer is used as the matrix [23]. As can be seen from Fig. 2.8, the immunological behaviour of various dilutions of reconstituted freeze-dried recombinant apo E 3 (one of the isoforms of apolipoprotein E) in relation to the frozen recombinant apo E~, the human VLDL (very low density lipoprotein) purified apo E 3 and fresh human serum samples, as determined using turbidimetric and ELISA methods, is similar. The stability of freeze-dried recombinant apo E 3 is acceptable, without any major alterations of its immunological reactivity. Freeze-dried serum certified for the determination of trace elements is used to check the accuracy and precision of analytical procedures for quantifying low levels of trace

Analvtical freeze-dpying

35

0.030 0.025 IZ >no I~D rv
99.998%) helium or nitrogen at a specified flow-rate, temperature and time (Fig. 4.5A). This extracts the volatile analytes from the sample matrix and transfers them to an ambient temperature trap (viz. a short, l/8-in i.d. packed column) containing from as few as one to as many as four different adsorbents. Following the purging and trapping step, volatiles are thermally desorbed (injected) onto the gas chromatographic column (Fig. 4.5B). This is accomplished by rapidly heating the He-swept trap to a high enough temperature in order to transfer the analytes in a narrow injection band in an attempt to simulate the effect of a direct syringe injection. Then, chromatographic separation and detection are performed as required. The final step in the analysis is the preparation of the sample concentrator for the next sample. This involves baking the adsorbent trap at a high temperature with flow through the trap in the reverse direction of the purge extraction flow (Fig. 4.5C). The baking step and the chromatographic separation are, for the most part, carried out simultaneously. It should be noted that most users of these dynamic techniques, and also equipment manufacturers, make no distinction between dynamic headspace (DHS) and purge and trap (PT). The difference, when considered [46], is based on bubbling of the auxiliary gas inside the sample in PT and sweeping it over the sample surface in DHS. All other

A) --L J",

B) " ~ WARM WRS"

(

~ TO " VENT

r-/

C)

)

' "-L, DESORBER

TO

0 17.5 7.6 117 11.8 288 99

Cg, ....

Ai

i _

Cg,2

A2

The partition coefficient can be derived from eqs (4.5) and (4.6), following rearrangement, and calculated from

A l ~ , - A2fl 2 K ...... A2 - A I

(4.7)

Table 4.2 lists the partition coefficients for selected compounds.

Multiple headspace. theory This HS mode uses a mathematical model developed by McAuliffe [58,59] that is applied in a stepwise manner. Step 1 corresponds to the end of the first equilibration process, when equilibrium between the sample in the vial and the headspace above it has been reached. Similarly as before, the thermodynamic equilibrium is described by the partition coefficient, but subscripts now include a number to identify the particular step. Thus:

K = C,I/C~I

(4.8)

m I = m~l + rngl

(4.9)

C~, = m~,/V,

(4. IO)

107

Solid s a m p l e treatments involving the removal o f volatile species

(4.11)

Cg I = mgl/Vg

Note that all concentrations in the previous equations are amounts per volume. Because equilibrium is followed by pressurization and sample transfer steps, and then by separation of the volatile compounds in the column, A l = const 9Cgl

(4.12)

It should be noted that, during sample transfer to the column, the total amount of analyte present in the vial's headspace is reduced by the amount transferred. The change is neglected here because the amount transferred is usually small relative to the total amount present in the vial. In the case of splitless sampling into an open-tubular (capillary) column, the total volume of gas taken from the headspace is about 50 gl. If a vial with a volume of 22 ml is assumed, 50% of which will be the headspace (/3 = 1), then the transferred aliquot will account for only 0.45% of the total headspace. Compared to the vented volume (see next step), this is significant. In the case of split sampling or when packed columns are used, the total volume of gas taken from the headspace is higher (about 0.5 ml, which accounts for 2.3% of the total headspace). Regardless of its volume, it is still reproducible and simply adds to the vented volume. Thus, at the end of step 1, the vial contains the respective headspace and sample volumes of ~ and V,; the amounts and concentrations of the analyte in these phases are mg~, m~, Cgz and C~. At this point, the pressure in the vial is Ph, which is higher than the atmospheric level. S t e p 1/2 is a transitional step corresponding to the venting step that follows the first headspace analysis (Fig. 4.4D). The gas phase in the vial is vented to the atmosphere; as a result, its pressure, Ph, is reduced to atmospheric or near-atmospheric level (P0). This step can be visualized as a gas expansion: the gas, having a volume V~, expands to V~z2 as its pressure is reduced to Po. Note that Po is not always identical with atmospheric pressure. Pressure release (venting) in the pressurized vial should be very rapid so as to avoid any change in the sample concentration (C~). Consequently, P0 may be slightly higher than atmospheric pressure as a result of the partial pressures of the matrix compound(s). This is particularly the case with aqueous samples when the pressure P0 in the vial is dictated by the saturated vapour pressure. Gas expansion during venting is instantaneous and, initially, only affects the gas phase. In other words, the amount and concentration of the analyte in the sample remains unchanged (the analyte has not time to repartition), so C~l/2 = C~ I =

m.~_

v,

(4.13)

Also, the amount mg~ originally present in V~ will now be present in the expanded volume lg~2, so the concentration of this expanded volume will be - m-gl-C g l l 2 = C s I - Vgl/2

and

(4.14)

108

Solid sample treatments involving the removal of volatile species

Cgl 2 < Cgl

(4.15)

From the basic gas laws, the relationship between steps 1 and 1/2 can be described as Ph" ~ = P,," Vg,2

~ .... P_o V~~/2

(4.16a)

(4.16 b)

Ph

We shall henceforward use p to refer to the ratio of the two pressures: (4.17)

p = po/ph

Solving eqs (4.11) and (4.14) for the respective volumes, and substituting the resulting expressions into eq. (4.16b) yields Cg 12/Cgl = p

(4.18)

The gas volume Vg~2 in this step consists of two parts, namely: one, V,.~, that is vented, and the other, Vg, which corresponds to the headspace volume above the sample in the vial" Vgl.2 = V,, + Vg

(4.19)

Similarly, the amount of analyte originally present in the vial headspace under equilibrium conditions, mg~, can be split into two parts, namely: one, mv~, 2, that is vented and the other, mg I 2, that remains in the vial headspace" mg I = m , i 2 + mgl 2

(4.20)

In this step, the analyte concentration in the headspace will be Cg~2; the same as established for the expanded volume [cf. eq. (4.14)]. Such a concentration can be expressed in terms of mgl, 2 and Vg (the vial's headspace volume)" Cgl 2 = mgl 2/Vg

(4.21)

Returning to the total amount m~ present in the vial in step 1, the following material balance can be written [of eqs (4.9) and (4.20)]: ml = mgl,2 + m,.i,2 S i n c e m,.I 2

(4.22)

is vented, the total amount of analyte remaining in the vial in this transition

step will be ml, 2 = msl + mgl2

(4.23)

Solid sample treatments involving the removal ()./'volatile species

109

In step 2, the needle is closed and the vial is re-equilibrated at the same temperature as in step 1. At equilibrium, the distribution coefficient will be the same as in step 1, which means that the ratio of the equilibrium concentrations will also be the same; however, the individual concentrations will now be different"

(4.24)

K = Cs2/Cg2

With these new concentrations, the respective amounts in the sample and gas phase at equilibrium will be m~2 and mg 2, respectively; because the volumes of sample and gas phase remain constant, C~2 = m,2/v~

(4.25)

Cg 2 = mg2/Vg

(4.26)

The total amount of analyte now present in the vial, m 2, will be the same as that at the end of the transition step, which, however, is less than m~ (the amount of sample present in step 1)" (4.27)

m 2 = ml, 2

Consequently, (4.28)

m 2 = ms2 + mg 2

Substituting eqs (4.23) and (4.28) into (4.27) yields m~2 +

mg 2 =

m, I +

mgl 2

(4.29)

Solving eqs (4.10), (4.21), (4.25) and (4.26) for the respective amounts allows the following material balance to be written: Cs2 " V s + Cg 2 "Vg = Csl . V s --F Cg I 2.Vg

(4.30)

Substituting/3- V~ for Vg in the previous equation gives

c,: + c ~ ./3 = C,, + c~,~ /3

(4.31)

By solving eqs (4.8), (4.24) and (4.18) for C,i, C,2 and Cgl 2, respectively, and substituting them into (4.31), one obtains

g : . K + c ~ ./3 = g , . K + g , o _Cgz _ K + b.p _ K/(~8 + p) C~ K + ~ K/(B + 1)

(4.32)


I I0

Substituting Cg~ for C~ 2 is legitimate because, at the end of step 2, the pressure in the vial will be the same as that at the end of step 1. If equilibrium is followed by pressurization and sample transfer, and by another GC analysis from the new headspace aliquot, then the peak area obtained will be A 2. By analogy with eq. (4.12), the following expression can be written: A2 - const 9C~2

(4.33)

Based on the proportionalities between the equilibrium headspace concentrations and the peak area [of. eqs (4.12) and (4.33)], the concentrations in eq. (4.32) can be substituted by the respective peak areas:

A2= K/([3 + p) A, K/([3 + I)

(4.34)

Further steps: if the MHS procedure is repeated several times, the peak area obtained in the i-th step can be described by the following relation:

A, _ (K/(fl + p)]~-

A,- X/(r + ii/

(4.35a)

or

A, _ (K/(r + 0))'-'

A, - \K)(fl + 1)

(4.35b)

Equation (4.35b) describes a geometric progression the quotient, Q', of which is Q, = K/([3 + p_)_

g/(13 + I)

(4.36)

The mathematical condition for a convergent geometric progression is that the value of its quotient, Q', is between - 1 and + 1. Since Po < Ph, P will always be less than unity. For this reason [of. eq. (4.36)], Q' will also be less than unity (between 0 and + 1). This fulfils the condition for a convergent geometric progression. Q' can be calculated from a regression analysis of the linearized form of eq. (4.35b): In A; - (i - 1). In Q' + In A I

(4.37)

The sum of the geometric progression expressed by eq. (4.35b) is

ilAi=l

.

.

.

.

AiQ, _ .

.

.

(4.38)

Substituting eq. (4.36) for Q' in eq. (4.38) yields

i I Ag = Ai

K/([3 + 1)

1- p

(4.39)

Solid sample treatnzents involving the removal of volatile species

111

This means that, if all three parameters (K,/3 and p) are known, the total peak area can also be calculated from a single area measurement. However, this is not recommended for two reasons: first, the parameter values are usually not readily available and their accurate determination would take at least as much time as the three or four consecutive steps of MHS; second, determining a single peak area provides no information about the existence of equilibrium conditions, which is a pre-requisite for MHS [60]. Recently, a new headspace mode was developed that allows complete or partial sampling of the vapour phase from a pre-equilibrated headspace vial, using a dual needle system. The theoretical treatment of this system has shown that the maximum mass (M,l,~,x) of an analyte in the gaseous phase (Mg) is generated when the gas-condensed phase ratio (Vg/Vc) in the equilibrated headspace vial is equal to the square root of the distribution coefficient [61].

Multiple headspace and solid samples The most important criticism concerning MHS is that it is very difficult to implement with solid samples [62]. Were this true, it would obviously severely restrict its applicability, but it is not. Proper conduct of an MHS procedure requires fulfilling the following five conditions: (a) the relationship between peak area and the concentration (amount) of analyte in the headspace should be linear throughout the concentration range of interest; (b) the coefficient of distribution of the analyte between the headspace and the sample should be constant throughout the range examined; (c) before the headspace is sampled, it should reach equilibrium with the sample in the vial; (d) pressure release (venting) from the pressurized vial against the atmosphere should be rapid enough to avoid any change in the analyte concentration in the sample during this step; and (e) p (= PJPh) should be the same in every MHS measurement step. Fulfilment of these conditions results in constancy of Q', which in turn results in a high correlation coefficient for the linear regression analysis of eq. (4.37) or, expressed differently, in a linear relationship between In A; and ( i - 1). In other words, if a linear relationship is found, the conditions are fulfilled, and vice versa. Condition (a) above is not exclusive to headspace analysis; in fact, it is a pre-requisite for quantitative analysis of any sample in gas chromatography. Essentially the same is true for condition (b). Condition (d) is primarily a design problem. Finally, constancy of p is assured by proper automation of the system (i.e. by exact repetition of the operational parameters) and by the fact that the calibration standard is carried through the same MHS steps as the sample itself. Therefore, the greatest problem is posed by the need to ensure equilibrium between the two phases in the vial. With liquid samples, the vial containing the two phases (gas and liquid) represents a partitioning system. Here, equilibration will sooner or later be accomplished and the time needed for it will depend primarily on the temperature and volume of the liquid. At increased temperatures, equilibration is faster than with larger volumes because, in the latter case, the analyte molecules take longer to diffuse in and out of the sample. Establishing the minimum time needed for equilibration in an unknown sample is one of the basic measurements recommended in headspace analysis [45,63].

112


With solid samples, equilibrium can only be reached under certain conditions or after specific changes. One major advantage of MHS is that it can be used as a preliminary approach to establish whether regression analysis will expose linearity in the In A; vs ( i - 1 ) relationship. This allows the analyst to determine whether the solid sample represents a partition system or an adsorption system. Typical solid samples representing a partition system include polymers heated above their glass transition point. These samples are amenable to application of the usual HS modes (e.g. the standard-addition technique). The standard can be added to the gas phase in the closed vial by injection through the septum, equilibrium being established by diffusion from both sides. If the regression analysis reveals the absence of linearity, then either some instrumental parameter (e.g. the sample temperature or equilibration time) will have to be adjusted or the system is not a partition but rather an adsorption system. If the solid sample represents an adsorption system, then the distribution of the volatile analyte(s) will be dictated by an adsorption coefficient. Usually, adsorption coefficients are a function of concentration or are constant over a short concentration range only. Obviously, such a non-linear system cannot be handled by the MHS extrapolation technique. There are, however, a number of alternatives for adapting the system to MHS or MHS-like determinations, three of which are described below. If the volatile analyte is present at a very low concentration and is mostly adsorbed on the sample surface, one can physically measure the peak area for the volatile analyte, step by step, until no further peak is found. Usually, only a few extraction steps are needed until the whole amount of analyte is depleted. In this case, the sum of peak areas will be proportional to the total amount of analyte present in the sample. Alternatively, one can convert the adsorption system into a partition system by adding a displacer to the sample in the vial headspace. One typical example is the determination of residual halocarbon solvent in decaffeinated instant coffee using an excess of water as displacer [45]. For analytes adsorbed on activated charcoal which is used in personal monitoring tubes - - benzyl alcohol is an effective choice for desorbing the volatile analyte [49,64]. Finally, one can use the "suspension approach" [65] to determine water in solid samples that would otherwise act as adsorption systems. The sample is suspended in a water-miscible dry polar solvent such as ethylene glycol monomethyl ester (EGME). The second solvent displaces water from the solid sample and dissolves it; if the water concentration in EGME is low enough (less than 5% v/v), then the peak area is linearly related to the concentration. With some solid samples, even the previous modifications fail and no apparent equilibrium is reached. Such is the case with coarse or bulky materials (e.g. large granules or solid pieces). Occasionally, the situation can be effectively improved by milling granules while cooling with dry ice, as in the determination of residual styrene monomer in large polystyrene granules [45]. It should be noted that the problem of very slow diffusion in and out of a bulky solid matrix makes any type of gas extraction impractical and the results dubious. In other words, these restrictions apply not only to the MHS approach but also to dynamic headspace processes in general.

l l3

Solid sample treatments involving the removal of volatile .species Purge and trap." theory

In a continuous gas extraction system such as purge and trap and dynamic headspace, the volatile analyte is continuously purged or removed from the sample by means of an inert gas system and the process can continue until the analyte is completely eliminated. The removed analyte is trapped from the gas stream; hence, at the end of the continuous gas extraction, the whole amount of analyte is available for determination using standard GC techniques, with no interference from the excess matrix compound. The efficiency of the separation process is controlled by the rate of diffusion of the analyte from the liquid or solid sample phase to the gaseous phase. An infinitesimal variation in concentration of the sample phase, dC~, can be described by the equation /CA dC.~ = - dt V

(4.40)

where K is the flux of analyte from the sample to the gas phase, expressed in mol/cm2 " s; A is the interface area, in cm2; and V is the solution volume, in millilitres. Based on Fick's first law and the Nernst approximation, eq. (4.40) can be rewritten as dC, C

.....

D h6

dt

(4.41 )

where D is the diffusion constant of the analyte, 6 the thickness of the diffusion layer and h the height of the sample in the vessel. Under the boundary condition that C~ - C, ~ at t - 0, integration of the previous equation yields a first-order kinetic-like function: C, = Q ' . e x p ( - b t )

(4.42)

where b = D/h6. The efficiency with which a given analyte will be removed can be estimated from the following ratio for an equivalent mass of analyte processed by both purge and trap (PT), and direct injection (DI): % Extraction = Area of analyte (by PT)/Area of analyte (by DI) The extraction efficiency for an analyte can be predicted using Henry's law at constant extraction volume, sample volume and extraction temperature [66]. The extraction of most analytes follows a first-order kinetics irrespective of the effects of hydrogen bonding or physical properties such as boiling point or water solubility [67].

4.3.4. Variables affecting performance This section deals not only with the variables pertaining to the headspace process (i.e. those affecting the removal of volatile species) but also with those involved in the passage of such species to the gas phase and the insertion of the compounds removed from the sample into the chromatographic column.

114

Solid sample treatments involving the removal of volatile species IZ~ O

u~

B)

0

o~

1

(:7}

C) 1

A) o e,)

=.2 o

I,-.

~3

Fig. 4.10. Chromatograms for solvent residues in pharmaceutical base material after equilibration at (A) 40~ (B) 70~ and (C) 80~ 1 benzene, 2 toluene, 3 o-xylene. All chromatograms were obtained by using a 15 m • 0.32 mm i.d. x 5 ~tm SBpTM-1 fused-silica column. (Reproduced with permission of Springer-Verlag.)

Variables common to all headspace modes

The partition coefficient is the driving force for removal of the volatile species to the gas phase in establishing equilibrium, attainment of which depends on the particular procedure. This coefficient depends on the analyte concentration: it decreases with increasing concentration but remains constant over a concentration range that depends on the particular type of compound. Thus, the range of virtual constancy for BTX (benzene, toluene and o-xylene) is 0.1-10 ~tg/ml; as a result, these compounds can only be determined reliably at concentrations below 10 ~tg/ml [68]. Temperature has a dramatic effect on the separation efficiency of conventional HS with both solid and liquid samples. The partition coefficient decreases slightly with increasing temperature. As a result, the headspace technique is more sensitive at high temperatures. Figure 4.10 illustrates the effect on equilibrium HS for liquid samples at three different temperatures [68]. Users tend to decrease the distribution coefficient


A)

I I5

140 130 >.n, LU >

0 o LU n~ a Z 0

120 110 100 90 80 70

n

60

0

50-

~

40

1 25

15

B)

J 1 35

1 45

1 55

1 65

75

SPARGE TEMPERATURE, ~ 200 180 >.-

160

w

140

O o uJ n," o

120

D

60

a.

40

O O

20

Z

o

100 80 -~---.--

0

1 25

15

c)

/ 1 35

1 45

1 55

I 65

75

SPARGE TEMPERATURE, ~ 180 o~ >.: n( LU > 0 o LU iY a Z 0

160 140 . . . . 120 100 80 60

n

40

0 o

20

J

15

1 25

1 35

1 45

1 55

I 65

75

SPARGE TEMPERATURE, ~

Fig. 4.11. Effect of the sparge temperature on compound recovery. (A) Gases. (B) Polar compounds. (C) Non-polar compounds. (Reproduced with permission of BPA International.)


116

A) CI3FC I

5.0

5.5

6.0

I

6.5

7.0

I

7.5

I

8.0

Acetone

i

8.5

I

9.0

I

9.5 TIME,s

B) CI3FC \

c)

5.0

I

5.5

I

6.0

I

6.5

I

7.0

1

7.5

1

8.0

Acetone

I

8.5

I

9.0

I

9.5 TIME,s

CI3FC ~N/j Acetone

5.0

i

5.5

i

6.0

i

6.5

i

7.0

i

7.5

i

8.0

i

8.5

i

9.0

i

9.5 TIME,s

Fig. 4.12. Extracted ion chromatograms for compounds 1-8 on the list of US EPA Method 524.2, rev. 4, at a sample sparge temperature of (A) 45~ (B) 55~ and (C) 65~ (Reproduced with permission of BPA International.)


117

(i.e. to raise the concentration of the target compound in the headspace) by increasing the thermostat temperature. However, the matrix compound should also be considered if it is volatile as its concentration in the headspace should not increase beyond a certain limit, particularly if its peak elutes from the column close to and/or before the analyte peak n which will have an unfavourable effect on the GC separation. Thus, when ethanol is determined from aqueous solutions (e.g. blood), samples are most often thermostatted at 60~ where the distribution coefficient for the alcohol is still high (about 550). Using a somewhat higher thermostat temperature would result in a lower distribution coefficient for ethanol (e.g. ca. 250 at 80~ At this temperature, however, the vapour pressure of water increases to an undesirably high level and its large peak, which emerges fairly close to that for ethanol, interferes with it. On the other hand, halocarbons have much higher vapour pressures at lower temperatures, so a near-unity distribution coefficient provides highly favourable conditions for their separation. The effect of temperature on PT depends strongly on the particular type of compound. Thus, a PT recovery study involving the analysis for VOCs using EPA Method 524.2, rev. 4 [69], which involved analysing replicate 5 ml aliquots of a 4.0 ng/ml standard at purge temperatures from ambient level (ca. 19~ to 65~ revealed the effect of temperature to depend on the specific analyte. As can be seen in Fig. 4.11, the increase in extraction efficiency with increasing temperature was more marked for the polar compounds, most of which were quantitatively recovered at 45~ Temperatures above this level resulted in little additional improvement in compound recovery, and, as can be seen from Fig. 4.12, also in decreased chromatographic resolution in some instances (primarily with polar compounds). The detrimental effect of increased temperatures arose from the increased amount of water that was transferred to the trap along with the compounds. The additional water occupied adsorptive sites in the trap that would otherwise have been taken by the target compounds. The additional water forced the target compounds to migrate further into the trap, thus having adverse effects on the desorption and chromatographic profiles [70-72]. The presence and nature of salts also affects to a lesser extent than temperature the partition coefficient, and hence the separation efficiency. The effect is illustrated in Fig. 4.13 for BTX at a constant concentration of the target salt; the effect of the salt concentration on K is shown in Table 4.3. It should be noted that the phase ratios in the vials for the liquid samples were kept constant at/3~ = 3 and/32 -- 1. The addition of solvents influences the separation of volatiles from samples by altering their partition coefficients. The sign of the effect depends on the relative polarity of the compounds and solvents involved. Table 4.4 illustrates the effect of the presence of an additional polar solvent such as an alcohol or acetone in an aqueous solution of BTX. As can be seen, the partition coefficient for the three analytes increases in the presence of a polar organic additive [e.g. 5% v/v alcohol (methanol, ethanol, n-propanol, n-butanol)] or acetone in the aqueous solution. Consequently, the additives aid dissolution of polarizable aromatics in water and ultimately decrease the sensitivity by a factor of 2-3. The sample volume has rather a different influence depending on the particular HS mode. For equilibrium HS, Ettre and Kolb introduced the concept "sample phase fraction", mathematically defined as q~ = VJVg, and showed that, provided this parameter

118


Fig. 4.13. Effect of salts on the partition coefficients of three analytes in aqueous solutions in a gas-water system. The salt concentration was 20% w/w. (Reproduced with permission of SpringerVerlag.)

is kept constant, the vial's volume has no influence on the results as the analyte concentration in the headspace will be almost constant except with low distribution coefficients [73]. In PT, the influence of the sample volume is dramatically affected by other variables including the sample temperature and total extraction volume. The above-mentioned study of VOC recovery using EPA Method 524.2, rev. 4 [69], revealed that changing the sample volume from 5 to 25 mi lowered the minimum detection limits (MDLs) for 93% of the 86 target analytes. However, MDLs were not reduced by a factor of 5 as might be expected; rather, the MDLs for all types of compounds (gaseous, polar and non-polar) were improved by a factor of only 2-3 relative to a 5 ml sample. Variables pertaining to purge and trap

The PT extraction efficiency depends essentially on two variables, viz. the total extraction volume and the sample temperature. The total extraction volume, or the total amount of purge gas that is passed through the sample during the extraction step (or over the sample in DHS) is determined by multiplying the flow-rate of the carrier gas by the time it is circulated. Since the earliest PT methods, the purge time has been specified to be 11 min at a flow-rate of 40 ml/min, which provides a total extraction volume of 440 ml. In theory, these flow-rates have been specified to optimize the extraction of volatiles (especially


119

TABLE 4.3 EFFECT OF SALTS ON PARTITION COEFFICIENTS IN AQUEOUS SOLUTIONS AT 40~ PHASE RATIOS:/31 = 3,/32 = 1 Partition coefficient Salt

Salt concentration (%, w/w)

Benzene

Toluene

o-Xylene

Na2SO 4

5 10 20 40

2.56 1.87 1.44 1.21

2.35 1.72 1.25 1.17

2.00 1.62 1.22 1.15

K2SO4

5 10 20 40

2.66 1.95 1.56 1.30

2.47 1.82 1.21 1.24

2.29 1.75 1.32 1.22

MgSO 4

5 10 20 40

2.72 2.05 1.65 1.35

2.52 1.97 1.32 1.29

2.31 1.78 1.37 1.20

NaCI

5 10 20 40

2.60 1.92 1.72 1.54

2.41 1.80 1.56 1.40

2.15 1.70 1.42 1.31

KCI

5 10 20 40

2.86 2.32 1.86 1.62

2.52 2.12 1.74 1.52

2.45 2.00 1.63 1.48

CaCI~

5 10 20 40

2.95 2.42 1.89 1.75

2.60 2.23 1.81 1.69

2.50 2.11 1.78 1.70

NaCH3COO

5 10 20 40

2.52 2.01 1.79 1.62

2.50 1.85 1.54 1.42

2.35 1.75 1.35 1.27

120


TABLE 4.4 PARTITION COEFFICIENTS IN AQUEOUS SOLUTIONS CONTAINING VARIOUS ORGANIC SOLVENTS AT A 5% CONCENTRATION AT 40~ Solvent

Methanol Ethanol Propanol Butanol Acetone

Partition coefficient Benzene

Toluene

o-Xylene

4.55 9.56 6.25 7.52 8.45

3.98 8.25 5.25 5.87 6.65

3.58 7.85 4.56 5.12 6.12

relatively non-polar analytes) from the sample matrix and trapping of the gaseous analyte on the concentrator's adsorbent trap. In practice, the purge flow is usually optimized over a series of standard analyses on a given system and is rarely, if ever, measured again. The effect of the purge flow-rate is becoming apparent as more polar analytes are being added to existing purge and trap methods. The analytes typically analysed by PT can be classified into the four categories of Fig. 4.14. The extraction of analytes that are difficult to purge can be improved by increasing the total extraction volume that is passed through the sample during the purge step. This can be done by increasing either the purge time at a constant flow-rate or the purge flow-rate. Additional purge flow, however, is likely to improve the extraction of inefficiently removed compounds only at the expense of the light gases. In general, over the specified purge time of 11 min, the extraction efficiency of all but the heaviest c o m p o u n d s - even those characterized as w e l l - b e h a v e d - decreases as a result of trap breakthrough across the total extraction volume range 200-600 ml. The new, more polar analytes added to EPA Method 542.4 (revised version 4) [73] have further challenged the purge efficiency tradeoff. In contrast to the analyte list in revision 3, the efficiency of these newly added analytes is generally improved over the 200-600 ml extraction volume range. Therefore, the trend towards reducing the purge time as a way of reducing the overall analysis time is likely to be reversed because of the addition of new purge-inefficient compounds. The breakthrough volumes for most volatile organics have been determined [74-76] and may constitute major reference material as the list of analytes processed using the purge and trap technique expands. Other steps in the PT process potentially influenced by temperature are adsorption and desorption. As noted in Section 4.3.3., the optimum temperature in the former depends both on the type of compound to be retained and on the trapping material used. As shown in Section 4.3.3, desorption performance is dependent upon the rate at which the trap is heated, the final trap temperature and the total desorb flow (i.e. the amount of gas that is passed through the trap). One other aspect of temperature to be considered is desorb preheat. Preheating the adsorbent trap immediately prior to desorption without

121


100J

WELL BEHAVED

a_" I--

HARD TO PURGE

75

o ii >ill

> o o

50 HARD TO TRAP

ILl

ISI Z

o o.

HARD TO PURGE AND TRAP

25

o o

I

I

I

I

I

I

I

2

4

6

8

10

12

14

PURGE TIME, min

Fig. 4.14. Different types of analyte behaviour towards purge and trap. (Reproduced with permission of Preston Publications.)

a carrier flow is usually redundant when rapid direct-resistance heating is used. However, the technique is occasionally useful to narrow the desorption bandwidth when the release of selected compounds from the trap is slow. Under these conditions, slowly released compounds spread out in the transfer gas stream and fail to be inserted into the column as a discrete plug. By heating the trap with no gas flow, the compounds can be released in a narrower band with reduced linear diffusion.

4.3.5. Calibration and analytical features Calibration

Calibration in headspace work is usually done by using one of four different techniques, namely: external-standard, internal-standard, standard-addition and vapour-phase calibration. External-standard calibration can be implemented by using either a simulated matrix or a pure matrix, the similarity between both being a function of the ease of preparing a simulated matrix closely resembling the pure matrix.

122


AA C

NS

] I

I0 150~

150~

5~

I

I

i

"l

0

70~

Fig. 4.15. Standard addition with a normalization standard (NS). Chromatogram obtained after headspace separation of 1.1% camphor (C) in a massage cream. (Reproduced with permission of Perkin-Elmer.) Single or multiple linear regression standard-addition calibration can be performed by using a single liquid or gas phase addition the latter being more appropriate for solid samples; however, it can also be implemented in conjunction with additional standards for normalization (see Fig. 4.15). Vapour-phase calibration (VPC) is based on the principle that the concentration of the volatile analyte in the gas phase can be determined by external-standard calibration. If the total amount present in the vial is known, the concentration in the sample phase at equilibrium is calculated from the difference. This technique, where the distribution of a volatile compound between two phases in a headspace vial is determined by using a pure vapour as reference, was originally implemented by Kolb using automated headspace equipment to determine distribution coefficients in gas-liquid [77] and gas-solid systems [78], and later by Schoene et al. [79] to determine solubility coefficients in vapours of both solid and liquid polymers. Although these investigations focused on

123


non-linear distribution as found both in concentrated solutions and in gas-solid adsorption systems, the same technique can obviously be applied equally well to dilute solutions, where the partition coefficient is constant and independent of the concentration. A description of the fundamentals of this technique can be found in a paper by Kolb et al. [80]. Sensitivity

The sensitivity of headspace-based methods can be modulated by altering a number of variables, namely: (a) The distribution coefficient and distribution ratio. Basically, the headspace sensitivity for a given concentration of a volatile compound in the sample depends on K and the phase ratio, /3 which includes the sample volume, V~. Whether an increased sample volume increases headspace sensitivity depends on K. Figure 4.16 illustrates the effect for dioxane (K = 642 at 60~ and cyclohexane (K = 0.05 at 60~ the t.D It)

,--/ J

1.0 ml

,

5.0 ml

/

/

O0 It)

/

d/ / tt3 03 03

I

0.0

I

5.0

I

10.0 MIN

K = 642

DIOXANE

I

15.0

I

0.0

I

5.0

t.iD t.iD 03 03

jV I

10.0

I

15.0

MIN

Fig. 4.16. Influence of K and fl values on the sensitivity of the headspace technique. (Reproduced with permission of Perkin-Elmer.)

Solid sample treatments involving the removal of volatile ,species

124 TABLE 4.5

HEADSPACE SENSITIVITY DEPENDING ON THE TEMPERATURE FUNCTION OF K Sensitivity = f(K) Solvent

40~

50~

60~

70~

80~

n-Hexane Toluene 2-Butanone Ethanol

0.16 2.8 166 1370

0.07 2.2 108 826

0.03 1.8 73 515

0.01 1.5 50 330

0 1.3 35 216

(b)

(c)

(d)

(e)

phase ratio, /3, was 21.3 and 3.46 at a sample volume (V~) of 1 and 5 ml, respectively. Also, based on their relationship to the area of the chromatographic peak, A ~ Q~ + fl). As noted in Section 4.3.4, the influence of the sample volume is only significant with low distribution coefficients. Temperature. The principal effect of this variable on sensitivity is exerted through the distribution coefficient. Table 4.5 shows the changes in K associated to temperature. As can be seen, the temperature can dramatically affect the sensitivity of HS methods. Addition of salts. The addition of an inert salt to an analyte solution can also affect the sensitivity of an HS method. The effect of the salt increases with increasing analyte solubility. The nature of the salt plays no crucial role in increasing the sensitivity. The effect is described in more detail in Section 4.3.4. Presence of polar solvents'. As noted earlier, the sensitivity of an HS method decreases when a polar solvent is added to an aqueous solution of low-polar compounds. Variables pertaining to the chromatographic and~or detection steps. Such variables are keys to both the selectivity and sensitivity of HS methods, and must be adjusted in accordance with the number, nature and concentration of the volatile compounds that are to be removed from the matrix. Additional factors such as cryofocusing or the injection mode also affect the performance of HS methods. A detailed discussion of their effect is beyond the scope of this book, however.

Precision The precision of HS-based methods relies strongly on strict control of the operating conditions, as well as on sample uniformity, the effect of which is more marked on solid than on liquid samples. The relative standard deviation of HS methods ranges from as low as 0.5% to as high as 50% [69,80,84,94].

Solid sample treatments involving the removal of volatile Lspecies

125

4.3.6. Applications The use of the headspace technique (particularly in its dynamic modes) for sampling volatile compounds in solids has by now reached most fields of industrial, environmental and social interest. In environmental analysis, the headspace technique (particularly in the purge and trap mode) has been extensively used in the determination of VOCs in soils, sediments and sludges [81-92]. In the industrial field, this separation technique has been used for purposes as diverse as the analysis of odour and taste problems in high-density polyethylene [93], contaminants in recycled high-impact polystyrene [94], benzene residues in recycled polyethylene terephthalate [95] and denture adhesives [96], residual toluene in medical plasters [97], and lipstick (for assignation of products to manufacturers) [98,99]. The analysis of natural compounds in foods is also assisted by the use of the purge and trap technique in methods for distinguishing strawberry varieties [100]; the aroma of unprocessed foods including gherkin [101], durian [102], garlic [103] and meat [104--107]; cheese [108,109] and other dairy products [110]; tobacco, tea and coffee [111,112]; and peanuts [112]. Food additives including sulphur dioxide [113,114] and .food contaminants such as VOCs [115-126], have been recovered by PT, particularly from table-ready foods. Animal [127,128] and plant tissues [129,130] have also been subjected to PT for separation of volatile compounds. The analysis of volatile organic compounds (VOCs) by PT is perhaps the most widely used method of trace analysis in environmental organic chemistry. The primary reasons for this massive use are that the method is applicable to a wide variety of analytes in virtually any type of sample matrix and that it is still unsurpassed in terms of sensitivity. The purge and trap technique has been adapted for the analysis of VOCs in a variety of matrices including human blood [131], consumer products (e.g. floor coverings and air deodorizers) [132], offshore waste gas in crude oil production [133], and on surfaces. The technique has also been used for the determination of residual solvents in pharmaceutical products [134], and is recommended for the analysis of bottled drinking water [135]. On the other hand, the equilibrium HS mode has been used for the determination of partition coefficients [80]. Good proof of the present maturity of headspace modes (especially PT) is the procedure developed by Supelco for the determination of BTX in soil and sediments [136] using a specially designed trap material. One uncommon application of PT is the monitoring of volatile hydride and methylated arsenic species produced by anaerobic microbes in gases released from sewage treatment facilities and municipal landfills [137]. Some commercially available units have been altered for specific applications; such is the case with an improved sparger for PT concentration that was used to determine volatile organics in landfill leachates and septic samples [138]. Losses of VOCs were minimized by using the same tube for sample collection, storage and sparging. Also, the technique of diffusive gradients in thin films [139] has been used as a step prior to PT for sampling sulphide species through a polyacrylamide hydrogel, accumulated sulphide being measured by PT followed by colorimetry [140]. The usefulness of HS methods is clearly reflected in their growing adoption by EPA [69]. Static and dynamic HS have been used in combination to identify aroma compounds

126

Solid sample treatments involving the removal of volatile species"

in yoghurt. Thus, overall, 91 compounds were identified by MS after HS-GC, nonen3-one being detected for the first time (at a concentration of 9 pg/kg) [141]. The performance of methods using various HS modes has been compared and found to depend on how strong the matrix-analyte bond is. Soil spiked with trichloroethylene and toluene was analysed using a gas chromatograph equipped with a PT concentrator that was found to be replaceable by a headspace unit in order to simplify the overall assembly. The headspace analysis of soil samples was found to be restricted by incomplete desorption of the contaminants in soil-water mixtures; this shortcoming, however, was effectively overcome by the addition of methanol. Henry's law constants for volatile organics in methanol must previously be determined if the method is to be applied to soils [142]. A comparison of the performance of static and dynamic (PT) headspace modes in the determination of nine VOCs in five different soils revealed poor PT recoveries in relation to those of static headspace (which ranged from 68 to 88%); the latter, however, required longer development times [143]. As stated above, most users of the headspace technique make no distinction between dynamic HS and PT. In one of the few publications that distinguished and compared these two HS modes, dynamic HS and PT were assessed as steps preceding highresolution GC-electron capture detection for the determination of nitrous oxide in sea water. The process was found to exhibit a first-order kinetics in both cases and the matrix to exert a significant effect that was proportional to the nitrous oxide concentration in bidistilled water, as well as in synthetic and natural sea water. As expected, PT provided better extraction recovery, sensitivity and limits of detection which fell in the picomole-per-millilitre range [46]. Based on available results, dynamic and static HS mode is complementary rather than competitive. The better choice in each case will depend on the sample-analyte interaction. Thus, the dynamic modes are better suited to semi-volatile compounds in water, while the static modes are to be preferred for solid and semi-solid samples. In any case, HS modes are not the panacea for separating volatiles from all types of sample; in fact, comparisons with other techniques have revealed HS to be no better choice in many cases particularly with strongly bound analytes. The primary US EPA-recommended methods VERVSW-846.5030 and 8260 A for measurement of VOCs in soils, which involves PT followed by GC-MS, only appears to be useful for measuring readily desorbed organic contaminants from soil pore spaces and external soil interfaces. It does not, however, measure contamination that has diffused into internal micropores of the soil matrix, as shown by Askari et al. [144]; thus, the purge and trap method measures only a small fraction of total soil contaminants, especially in long-contaminated soils, where about 90-99% of contamination is within the soil matrix. These authors compared three methods for determining VOCs in aged field samples, viz. purge and trap, methanol immersion and hot methanol extraction. As can be seen from Table 4.6, hot solvent extraction proved much more effective than the EPAapproved PT method. The results also reflect the inefficacy of PT as a method for assessing vapour extraction remediation technology and suggest that EPA should revise the use of PT methodology for measuring VOC concentrations in soils as the efficiency of the methods is strongly dependent upon analyte-matrix interactions.

TABLE 4.6 COMPARISON OF THREE METHODS FOR MEASURING VOC IN SOILS

Soil type (component)

Compounds

Purge and trap1.", m&lk&

Kentucky (clay)

Trichloroethylene

100

Louisiana (silty loam)

cis-I, I-Dichloroethylene

3070 i 35 1

Trichloroethylene

5900 i 1210

Methylene chloride

240

Benzene

2+1

Toluene

190

Chloroform

2*0

Florida (silty, fine to very fine sand)

Methanol ~mrners~on'"'. m&k

Hot methanol extraction'"', mg'kg

* 4359'h1 85 000 * 13 11Shl 530 * 31'h1

* 2082Ih' 121 700 * 11 719'h1 630 * 58Ih1 150 * 29Ih1 270 * 37Ih' 130 * 2Sh'

* 57

* 62 * 41

','I Average of three soil sample measurements Ih' Significantly higher mean than purge and trap at 95% confidence level "'Significantly higher mean than purge and trap at 90% confidence level

26 000

110 i 5Ih' 240 k 1l1'I l I0

5

51h1

41 700

128


4.4. ANALYTICAL PERVAPORATION

4.4.1. Introduction Analytical pervaporation emerged in the late 1980s as a non-chromatographic continuous separation technique advantageously competing with other membrane-based separation techniques thanks to the absence of sample-membrane contact, which makes it useful for dirty, aggressive and particle-containing samples [145-148]. The later integration of steps of the analytical process other than separation (viz. derivatizing chemical [149,150] and biochemical reactions either preceding [151,152] or following [153,154] the pervaporation step) increased its potential and miniaturizability. Finally, its use as a tool for treating solid samples has further expanded its scope. One of the most salient, favourable aspects of pervaporation is its ability to integrate many steps of the analytical process, thereby facilitating miniaturization. On the other hand, the greatest shortcomings of this technique are its still incipient development and the resulting absence of commercially available equipment to implement it.

4.4.2. Principles of pervaporation Industrial pervaporation

Industrial pervaporation [155-157] selectively separates a liquid feed mixture by partial vaporization through a non-porous polymer membrane. The separation is not based on relative volatilities, as in the case of distillation or evaporation, but rather on the relative rates of permeation through the membrane. The mixture is circulated through the membrane and the permeating component diffuses through it. A vacuum (vacuum pervaporation) or sweeping gas (sweeping-gas pervaporation) is applied to the membrane on the permeate side and the permeating component desorbs from the membrane as a vapour that can be collected or released, as required. The driving force for the separation is the chemical potential on both sides of the membrane. The permeating component is transported through the membrane because its partial pressure on the permeate side is lower than that in the saturated vapour [158]. Separation from mixtures is achieved because the membrane transports one component more readily than the others, even if the driving forces are equal. The effectiveness of pervaporation is measured by two parameters, namely: flux, which determines the rate of permeation; and selectivity, which measures the separation efficiency of the membrane (controlled by the intrinsic properties of the polymer used to construct it). The coupling of fluxes affecting the permeability of a mixture component can be divided into two parts, namely: a thermodynamic part expressed as solubility, and a kinetic part expressed as diffusivity. In the thermodynamic part, the concentration change of one component in the membrane due to the presence of another is caused by mutual interactions between the permeates in the membrane in addition to interactions between the individual components and the membrane material. On the other hand, kinetic coupling arises from the dependence of the concentration on the diffusion coefficients of the permeates in the polymers [ 155].

129


For a binary mixture of components A and B, the flux can be expressed for the entire permeate (Jr, total flux) or each component (J,, and JB, the flux of component A and B, respectively), having dimensions of mass/(area • time). The flux can be calculated provided the mass of the permeating component, the membrane area and the time of measurement are known. To this end, the following expression can be used: Ji = - Li Au_,.ri

(4.43)

I where L; is the phenomenological diffusion coefficient, At.ti the chemical potential driving force across the membrane and l the membrane thickness. The selectivity of pervaporation is given by the ratio of the mass fractions of components A and B for the permeate and feed. In the case of selective permeation of component A, the following equation applies: a A = yA-/yt3.. XA/XB

(4.44)

where x A and x~ are the mass fractions of components A and B, respectively, in the feed, and YA and Yl3 those in the permeate. A value greater than unity indicates selective permeation of A over B, whereas a value smaller than unity indicates selective permeation of B over A. The selectivity is dimensionless and occasionally described as an enrichment factor/3 that is the ratio of the concentration of one component on the permeate to that in the feed [ 158]. The process is favoured by increased temperatures, which result in a vapour pressure difference that increases the permeability of the substances through the membrane by allowing the diffusion of permeated molecules and decreasing interactions between permeates. The morphology of the membrane (rubber, glassy polymer) also influences the separation efficiency. Although pervaporation appeared at the beginning of the 20th century, research aimed at expanding industrial applications of this technique did not start until the 1970s, where the world energy crisis promoted the development of three different separation approaches, namely: (a) the dehydration of aqueous-organic mixtures by using water-selective hydrophilic membranes (e.g. the dehydration of ethanol produced by fermentation of sugar in refineries); (b) the permeation of organics from an aqueous-organic mixture using a hydrophobic membrane for purposes such as solvent recovery, hazardous waste treatment, water purification or beverage processing; and (c) the permeation of specific organic compounds from an anhydrous mixture (e.g. aromatics-paraffin, branched hydrocarbonsparaffin, olefins-paraffin, isomer mixtures), which is unfeasible with currently available membranes. Pervaporation surpasses conventional industrial separation in several respects; thus, it makes more efficient use of energy, allows the ready separation of azeotropic mixtures and dehydration of multicomponent mixtures, avoids contamination of the product with entrained compounds and the environmental pollution usually resulting from treatment of entrained substances, uses little space and is easy to implement and install on-site as the pervaporator is skid-mounted [155].

130


Analytical pervaporation Analytical pervaporation is the process by which volatile substances in a heated donor phase evaporate and diffuse through a porous hydrophobic membrane, the vapour condensing on the surface of a cool acceptor fluid on the other side of the membrane. Surface tension forces withhold the fluids from the pores and prevent direct contact between them. A temperature difference that results in a vapour pressure difference across the membrane provides a strong driving force for the separation, which also occurs in the absence of a temperature gradient. Evaporation will occur at the sample surface if the vapour pressure exceeds that at the acceptor surface. One important feature of pervaporation modules used for analytical purposes is the air gap between the donor phase and the hydrophobic membrane, which avoids any contact between them and reduces the problems associated with fouling of the membrane. The mechanism of transport by pervaporation can be described in the light of the sample diffusion model [159], which comprises the following steps: (a) evaporation of the analyte into the air gap; (b) sorption into the membrane on the sample side; (c) diffusion of the sorbed component through the polymer matrix; and (d) desorption into a liquid or gas phase on the acceptor side. The last three steps are also included in industrial pervaporation processes. Analytical pervaporation is a very mild process that can be operated at the required temperature and needs no high pressure or cross-flow velocity, and no additional chemicals. Because of its short life, the theoretical principles of analytical pervaporation have not yet been established except for liquid samples and gas-phase acceptors [160]; there is, however, research in progress on solid and liquid samples processed with both liquid and gaseous acceptors [ 161 ].

4.4.3. The analytical pervaporator and auxiliary units So far, laboratory-scale pervaporators have been user-designed and built. Although pervaporation can be applied to liquid, solid and slurry samples, the basic separation unit is identical whichever the sample type, the sole difference as regards equipment requirements being the use of appropriate accessory units. An analytical pervaporator consists of two essential parts, namely: the body of the separation module (including the devices for hindering gas losses) and the membrane.

Pervaporator body Basically, an analytical pervaporator consists of the elements shown in Fig. 4.17A, namely: an upper acceptor chamber (a) with inlets and outlets through which the acceptor stream is circulated and in which the gaseous analyte (or its reaction product if the analyte is not volatile) is collected; a lower, donor chamber (d) that contains the solid sample or through which the feed stream of liquid or slurry sample is circulated; a thin (ca. 1 mm) membrane support (b) made of polytetrafluoroethylene (PTFE) or metal; and spacers (c) of variable thickness (2-10 mm) that can be placed below or above the membrane support in order to increase the volumes of the corresponding chambers.


A)

131

B)

(a) (i)

~

(g)

(a) (b) ~ ~ ) (c)

(b)

(i)

(d) (d) .

.

.

.

.

.

.

.

.

.

.

.

(e) Fig. 4.17. (A) Parts of a conventional pervaporation module: (a) acceptor chamber; (b) membrane support; (c) spacer; (d) donor-sample chamber; (e) aluminium supports; (f) and (g) rods for screwing and aligning the module, respectively; (h) connectors; (i) screws. (Reproduced with permission of Wiley & Sons.) (B) Most significant parts of a hexagonal pervaporation module: (j) membrane. (Reproduced with permission of Elsevier.) The whole module can be made of methacrylate, which enables continuous checking during experiments. Some Perspex pervaporators have also been reported [162]. The upper and lower chamber, and the membrane support, are aligned by inserting metal rods in the four orifices (g); closer contact is achieved by screwing them with four screws (f) between two aluminium plates (e). The temperature is adjusted either by circulating a fluid at an appropriate temperature [163], by immersing the module in a thermostatted water bath [150], by inserting an electronic temperature controller [164] or by using focused microwaves [28]. Usually, only the lower chamber is heated and no temperature control system is used for the upper chamber. The efficiency of the pervaporation step can be boosted by cooling the acceptor chamber; however, special precautions must be adopted as the temperature gradient may result in condensation of water vapour present in the air gap upon contact with the sample side of the membrane, thus decreasing its active surface. The hexagonally shaped unit of Fig. 4.17B was designed with the aim of decreasing void volumes and increasing throughput through faster removal of both the sample and acceptor solutions containing the pervaporated species from the donor and acceptor chamber, respectively.

132


The analytical pervaporator can be used in combination with a flow-injection manifold, either in the upper chamber when the pervaporated species must be derivatized for adaptation to the detector and/or in the lower chamber for the pervaporation of analytes from liquid samples or slurries. Alterations of either the auxiliary dynamic manifold or the pervaporator itself are required when the pervaporation step is assisted by focused microwaves, the separation step assists in the continuous monitoring of an evolving system, untreated solid samples are used or pervaporation is integrated with detection. The module used for microwave-assisted pervaporation [28] is similar to that shown in Fig. 4.17, but smaller so that it can be accommodated inside the microwave vessel. Since no metal parts are admitted in the microwave chamber, the cell is constructed entirely in PTFE/methacrylate and the screws in PEEK" - poly(ether-ether ketone). When the pervaporation unit is used for the continuous monitoring of a fermentation process with a view to determining analytes in samples containing suspended particles, or in slurries, the module is altered by increasing the diameter of both channels (inlet and outlet) of the donor chamber, which might otherwise be clogged. For solid samples, a septum is placed at the inlet of the lower chamber through which the reagents, if necessary, are injected with a syringe furnished with a hypodermic needle. The outlet of the lower chamber is shut with a screw in order to avoid leakage and the resulting loss of analytes. When separation and detection of volatile species occur simultaneously, a hole is drilled at the centre of the upper chamber top of the pervaporation cell in order to accommodate the sensor [149,165] with the aid of appropriate adaptors. This module can be used with both liquid [149,152] and solid samples [165]. The flow of the acceptor stream is stopped during measurement so as to allow the accumulation of the analyte released from the matrix during the process and hence to increase the sensitivity [166]. Membranes

As a rule, permeability in glassy polymers (e.g. cellulose) is lower than in rubbery polymers (e.g. polydimethylsiloxane, PDMS); on the other hand, selectivity is dictated by the molecular dimensions of the permeating species [167]. The polymers used as membranes in analytical pervaporation are similar to those employed for gas separation and possess a dense, non-porous macroscopic structure. The difference between the two lies in the transport mechanism and arises mainly from a large affinity difference between the permeating molecules and the polymer membrane. The membranes used for analytical pervaporation are hydrophobic membranes of the types usually employed in ultrafiltration and gas-diffusion processes. In practice, PTFE is the most frequently used membrane material, followed by hydrophobic polyvinylidenefluoride (PVDF). Ultrafiltration membranes are very thin, which, in combination with the large surface area of both the donor and acceptor chamber, leads to their easy bending. This results in changes in the flux of the permeating component through an altered membrane area and hence in changes in the efficiency of the process. As a result, membranes must be replaced fairly often. Because of their thickness, gas-diffusion membranes are not so easily bent, so the same membrane can be used over long periods. The pore size of the

Solid sample treatments involving the removal ()./volatile species

133

P R2

................................................ AS

DS S

'

.L .

. J. .

fL _ _ _ I 1

'..I -ll'~

~

--,\~_/\ ^

9 PM

. _ ,.A "

"'"

RC2

~

W2

\

L/

Fig. 4.18. Continuous-discrete approach to implementing analytical pervaporation of solid samples. The dotted line corresponds to a potential derivatization reaction of the pervaporated species and the dashed lines represent the continuous manifold used for automatic insertion of liquid samples. P peristaltic pump, AS acceptor stream, IV injection valve, SV switching valve for changing between continuous and discrete insertion of sample into the pervaporator, R reagent, DS donor-sample stream, S sample, RC reaction coil, PM pervaporation module, M membrane, D detector, W waste.

membrane (or its directly proportional thickness) influences the efficiency of the process and hence its sensitivity. This facilitates application to samples with variable analyte concentrations.

Auxiliary maniJbld Figure 4.18 depicts a typical flow-injection pervaporation assembly. Solid lines represent fixed parts and the dashed lines, parts included when liquid samples are to be processed. For solids, an appropriate amount of sample is weighed in the donor chamber, which is then connected to the rest of the pervaporator. The reagents, when necessary, are injected through the septum (at the inlet of the donor chamber) or septa (at both the inlet and outlet of the donor chamber) when several separate reagents are required. Then, the donor chamber is heated and the analytes pervaporate through the membrane and are accepted by the fluid on the other side of the membrane. The manifold into which the upper chamber is inserted does not depend on the initial state of the sample, but only on the characteristics of the pervaporated analytes, the type of detector used and its position along the manifold. Depending on the particular type of detector used, auxiliary channels will have to be included to bring the pervaporated species into contact with appropriate reagents in order to obtain products to which the detector will respond. Integrated detection and pervaporation requires altering the pervaporator but simplifies the overall manifold. As shown below, preconcentration units, solid-phase reactors (mainly enzyme reactors) and various other devices can also be connected in-line in the manifold when required.

134


Pervaporation modes

The qualifiers "continuous" and "discrete" as applied to pervaporation refer to different aspects of the process. In fact, analytical pervaporation is a continuous technique because, while the sample is in the separation module, mass transfer between the phases is continuous until equilibrium is reached. "Continuous" also refers to the way the sample is inserted into the dynamic manifold for transfer to the pervaporator. When the samples to be treated are liquids or slurries, the overall manifold to be used is one such as that of Fig. 4.18 (dashed lines included). The sample can be continuously aspirated and mixed with the reagent(s) if required (continuous sample insertion). "Discrete" sample insertion is done by injecting a liquid sample, either via an injection valve in the manifold (and followed by transfer to the pervaporator) or by using a syringe furnished with a hypodermic needle [directly into the lower (donor) chamber of the separation module when no dynamic manifold is connected to the lower chamber]. In any case, the sample reaches the lower chamber and the volatile analyte (or its reaction product) evaporates, diffuses across the membrane and is accepted in the upper chamber by a dynamic or static fluid that drives it continuously or intermittently, respectively, to the detector except when separation and detection are integrated. The flow of donor (sample) solution and acceptor fluid (lower and upper chamber, respectively) can be continuous or stopped at any time during the separation step in order to increase the efficiency of the process. With solid samples, the lower chamber always operates in a discrete manner since, as noted earlier, the sample is weighed in it. In this case, the separation module is also used as a leaching reaction vessel, which also can incorporate the detection step, thus operating as a mini-autoanalyser integrating all of the steps of the analytical process through an appropriate computer connected to the detector via a passive interface [165].

4.4.4. Pervaporation efficiency The efficiency of a given pervaporation process must be evaluated in order to act on the overall system so as to either boost or reduce mass transfer to the acceptor chamber as required. Such evaluation can be done either in (a) relative terms by comparing signals provided under different conditions by the analyte (or its reaction product), previously collected in the upper chamber and driven to the detector, or in (b) absolute terms by comparing the signal obtained under the working conditions with that corresponding to 100% mass transfer. The relative procedure is typically used in optimization experiments in order to accommodate the sensitivity (usually by maximizing it), but also to ensure the best possible conditions for derivatization reactions (prior and/or subsequent to pervaporation) and dispersion along the continuous system, among others. No special alterations of the manifold other than those resulting from the optimization process are required in this case. The absolute procedure requires an auxiliary channel in the upper subsystem, through which the acceptor solution is also passed, furnished with an auxiliary injection valve (AIV in Fig. 4.19) of the same characteristics as the main injection valve (MIV), which is used to inject the sample for analyte separation and monitoring. Two sequential sample

135


AUX AS

(~

lV ID I

PM

DS ~MIV

v

W2

Wl

Fig. 4.19. Simplified scheme of a hydrodynamic manifold for evaluation ofpervaporation efficiency. Note that, when a derivatization reaction is required prior to or after analyte separation, one or more additional channels for the reagent solution(s) must be included in the donor and acceptor stream, respectively. AIV auxiliary injection valve, MIV main injection valve, PM pervaporation module, m membrane, a merging point, D detector, W waste, AUX auxiliary channel containing acceptor solution, S sample, AS acceptor solution, DS donor solution. (Reproduced with permission of Wiley & Sons.)

injections the sequence is not influential from both valves are needed to evaluate the efficiency of the mass-transfer process. First, a volume of sample containing a given analyte concentration is injected via MIV; the analyte evaporates to the air gap, diffuses across the membrane, and is absorbed by the acceptor stream and driven to the detector. The signal produced is proportional to the amount of analyte transferred from the donor to the acceptor stream. Second, an identical volume of the same sample is injected through AIV and driven to the detector. A signal corresponding to the whole amount of injected analyte is obtained. The difference between the two signals provides the amount of analyte transferred to the separation process relative to that in the sample. Note that the dispersion undergone by the monitored species in the two channels is different, so the two must be compared in terms of the areas of the transient signals (representative of the total amount of monitored species that has passed by the detection point) rather than their heights, which are representative of the concentration. Obviously, the comparison is not valid if the flow-rate in the upper manifold changes during the process [168].

4.4.5. Variables influencing analytical pervaporation Most often, the aim of manipulating the variables that influence a method is improving its efficiency, both by lowering the limit of determination and by obtaining a steeper slope for the calibration curve. When the concentration of the target analyte in the sample


136

varies widely, a real or pseudo dilution step is occasionally mandatory in order to fit the analyte concentration within the linear range of the calibration curve. Some ways of changing the mass transfer through the membrane in analytical pervaporation are obvious and can be inferred from the nature of the separation process; others, however, are sophisticated or ingenious approaches. The variables can be characteristic of either the donor or the acceptor chamber (or manifold) of the system.

Variables pertaining to the donor chamber An increase in temperature in the donor chamber increases the vapour pressure of the analyte and boosts mass transfer across the membrane. The usual way of heating this chamber is by immersing it into a water bath. When pervaporation is assisted by microwaves focused on the donor chamber, higher temperatures can be reached with closer control [28]. Different working temperatures result in calibration curves that encompass different analyte concentration ranges (see Fig. 4.20), which can be of interest when the concentration of the target species in the sample ranges over wide bounds. Sample agitation is strongly influential on pervaporation. Magnetic stirring facilitates the removal of gases from the donor chamber, thus accelerating attainment of the separation equilibrium. Ultrasounds have a similar, but stronger, effect compared to magnetic stirring; however, this type of energy favours leakage and hence losses of the gas phase. The presence of chemically inert beads" of a suitable size in the donor chamber boosts the transfer of volatiles to the air gap, both with liquid and with solid samples. In the 1.6 1.4ILl

(O Z

,,~

1.2-

I I

1.0-

rn

n,"

O

0O

0.8-

J

J

0.60.4-

7

,.u-"J

J

J

J

O-'-"'---

0.20.0 0

I

I

I

I

4

8

10

15

20

[ETHANOL, %]

Fig. 4.20. Calibration curves for ethanol obtained at a pervaporation time of (1-1) 8 min and (O) 4 min.


137

former case, the time the sample remains in the donor chamber is increased by effect of the beads creating a tortuous path for the liquid [159]. With solids, the beads separate the particles, thus creating a larger surface for transfer of the gas to the air gap. The volume of the air gap over the sample is crucial for efficient pervaporation as it contains a large enough amount of evaporated species to force passage through the membrane by gradient. The smaller the air gap is, the smaller is the amount of analyte required to start mass transfer through the membrane. The best and most controllable way of decreasing the air gap volume in the case of liquid samples (viz. with a continuous manifold connected to the lower chamber) is by lengthening the waste line. This channel creates a pressure in the lower chamber that is proportional to its length. By increasing the length, the overpressure created forces up the level in the donor chamber, thereby decreasing the amount of evaporated analyte needed to establish equilibrium in the gas phase, which results in faster mass transfer through the membrane. Thus, the efficiency obtained by using a waste tube of 280 cm rather than one of 20 cm (both of the same inner diameter) is 14 times higher [147]. Reducing the air gap space also increases the likelihood of sample-membrane contact, so a compromise between sensitivity and selectivity must be adopted. With liquid samples, the.flow-rate strongly influences the pervaporation process. The lower the flow-rate is, the longer will be the residence time in the donor chamber of a given volume of sample and the closer to equilibrium will be the analyte mass transfer. The flow can be stopped when the sample is in the donor chamber to allow steady-state mass transfer. This situation is not desirable because it lengthens the separation unduly. Instead, a fresh sample can be passed through the donor chamber as often as required provided the volume of sample available is not limited. The concentration of reagents (pH included) can have dramatic effects on pervaporation if the reagents take part in the reaction that produces the volatile species. In any case, mass transfer can be modulated as required by altering such a concentration.

Variables pertaining to the acceptor chamber or auxiliary mani[bld Mass transfer through the membrane can be adjusted by changing two major variables of the acceptor (upper) chamber. Thus, the .flow-rate of the acceptor fluid is a key to improved pervaporation; this can be achieved through a displacement in the mass transfer equilibrium resulting from the continual use of fresh acceptor fluid w which maximizes the concentration gradient between both chambers. Keeping the fluid static facilitates attainment of an equilibrium or near-equilibrium state for mass transfer. There are two ways of keeping the acceptor fluid static during pervaporation. One is by stopping the propulsion unit for this stream. This entails using separate propulsion units for the lower and upper streams (if both are required), and disturbs the detector baseline through the start-and-stop sequence, which in turn causes part of the fluid to return to the propulsion unit. These shortcomings can be avoided by using the approach illustrated in Fig. 4.21A, where the upper chamber of the separation module is accommodated in the loop of an injection valve. In the filling position, the acceptor stream passes through the bypass to the detector at the preset flow-rate. After the predetermined collection time has elapsed, the valve is switched to its load position and the stream


138

A) IV2

AS

P,

-= W2

IV1 -= Wl

P2

B)

IEC P1 AS

[~

E

PR PM

___

IV

DS Wl

c)

INLET

1

l

OUTLET

Fig. 4.21. Different ways of improving pervaporation efficiency. (A) By halting the flow in the acceptor chamber without disrupting the overall dynamic system. (B) By on-line retention of transferred volatile species and elution in the opposite direction. (C) By use of a packed flow-cell to integrate reaction and detection. E eluent, IEC ion-exchange column, PR preconcentration, EL elution. (For other abbreviations, see previous figures.) (Reproduced with permission of Wiley & Sons.)


139

passed through the loop to sweep its contents to the detector. In this way, a situation as close to mass transfer equilibrium as required can be established without disturbing the detector baseline. Preconcentration ofpervaporated analytes is the most effective way of favouring mass transfer. The equilibrium between phases can be efficiently displaced by continuous removal of the transferred species from the upper chamber, followed by concentration (either in a minicolumn placed before the detector or in the flow-cell itself). A minicolumn packed with an ion-exchange sorbent can be placed between the pervaporation module and the detector, either in the transport tubing or, better, in the loop of an injection valve (Fig. 4.21B). The latter approach allows the retention and elution steps to take place in opposite directions in the column, thus avoiding increased compaction and hence an increased pressure in the dynamic system [169]. By retaining the pervaporated species in the detection zone, the previous two steps can be integrated and the kinetics of the separation process monitored as the volatile species will reach the detector at the same rate as they cross the membrane (Fig. 4.21C). Obviously, this approach is only viable with non-destructive detectors. Both approaches (Figs 4.21B and 4.21C) allow fresh portions of the acceptor fluid to be brought into contact with the membrane in such a way that equilibrium of the separation process is never reached. Characteristics of the membrane such as pore size and, especially, thickness, have a strong influence on pervaporation. A comparison of three PTFE membranes of pore size 0.5, 2.0 and 5.0 ~m, and thickness 0.16, 0.18 and 0.20 mm, respectively, in terms of permeability to hydrogen cyanide revealed the membrane of 2.0 lam pore size and 0.18 mm thickness to be the most permeable [162]. The membrane of 5.0 ~tm pore size was supposed to exhibit the highest permeability; however, its relatively large pores only allowed better diffusion of the larger molecular gases and not of the smaller ones such as HCN. In this situation, the effects of additional membrane properties including thickness and pore density must be considered. Permeability is inversely proportional to the thickness of the membrane and directly proportional to its pore density. An increased membrane pore size can be expected to require a decreased pore density to ensure acceptable mechanical stability. As a result, the overall area of the pores through which gas diffusion takes place could even decrease with increasing pore size and lead to decreased permeability. 4.4.6. The physics of analytical pervaporation: kinetics of the mass transfer process Monitoring mass transfer kinetics is of paramount importance in optimization experiments with a view to both identifying the factors influencing the process and establishing an appropriate pervaporation time to ensure a high throughput without significantly detracting from sensitivity. The kinetics of the pervaporation process can be monitored in two ways, namely: (a) by integrating pervaporation and detection; and (b) by integrating a retention step with detection (following pervaporation). Continuous monitoring of mass transfer across the membrane can be accomplished by placing the active surface of a sensor opposite the membrane (see Fig. 4.22A). In this

140


A)

P AS

TO TRANSDUCER

J

t-

m INLET

PM

o o9

z w

OUTLET

MEMBRANE

B) E R

_

~ ~

_

m

RC

r. W PACKEDFLOWCELL

Fig. 4.22. Monitoring of pervaporation kinetics. (A) By integrating separation and detection. (B) By coupling pervaporation with a retention step integrated with detection. For abbreviations, see previous figures. (Reproduced with permission of Wiley & Sons.)

way, a rising signal is obtained that reflects the enrichment of the static acceptor solution with the pervaporated species. The signal evolves to a plateau when the partitioning equilibrium of the analyte between the sample, air gap and acceptor fluid is reached. Electroanalytical (potentiometric, voltammetric) probes and fibre optic-assisted optical detectors have been used for this purpose. A flow-cell packed with a suitable support (e.g. an ion-exchanger or sorption material) accommodated in a non-destructive detector can be used for continuous retention of the pervaporated species or its reaction product (Fig. 4.22B). In this way, the content of the upper chamber is continuously removed and driven to the detector, and the fresh acceptor stream that reaches the chamber displaces the mass transfer equilibrium, thus increasing the efficiency of the pervaporation process. A rising recording is thus obtained, the slope of which evolves to a plateau but never becomes fiat since the concentration of the volatile species in the lower chamber becomes increasingly lower but is never zero. After the measurement interval the duration of which depends on the quantitativeness to be achieved has elapsed, a switching valve (SV) located prior to the detector allows the acceptor stream to be replaced with an eluting solution that removes the retained species and drives them to waste. On switching the valve again, the flow of the acceptor solution through the detector is restarted, the baseline re-established and the system made ready for a fresh sample.


141

4.4.7. Analytical features of pervaporation methods This section deals with the basic analytical properties (sensitivity, selectivity and precision) of pervaporation-based methods.

Sensitivity The sensitivity of a method involving pervaporation essentially depends on the efficiency of the separation step. The efficiency of pervaporation can be adjusted in order to fit the signal provided by the analyte or its reaction product to the linear portion of the calibration curve for a given method, thus avoiding the need for a dilution or concentration step. A number of approaches to increasing and decreasing pervaporation efficiency exist. The analytical signal can be optimized for quantitation by increasing the temperature of the donor chamber, stirring the sample, using chemically inert beads, employing optimal reagent concentrations, increasing the pervaporation time, using an efficient procedure for concentrating the volatile species and increasing the amount (weight or volume) of sample used. When the concentrations of the target analytes in the sample exceed the upper limit of the linear range of the calibration curve, a dilution or pseudo dilution step is mandatory in order to fit the unknown concentration to this portion of the calibration curve, and hence to increase the precision of measurements. A number of approaches can be used in addition to the usual previous dilution of the sample or the weighing of a smaller amount to avoid the errors involved in a dilution step and in the weighing of small amounts of a solid. Such approaches involve (a) the use of a lower pervaporation temperature; (b) changing the chemical conditions to a less favourable situation, if a derivatization reaction is required, so as to reduce the yield of the volatile or monitored species (with the reaction preceding or following pervaporation, respectively); (c) expanding the air gap between the sample and membrane by intercalating as many spacers as required; (d) using a thicker membrane; (e) using a smaller loop for the injection valve (if the sample is a liquid); or (f) rising the flow-rate of the donor and/or acceptor stream.

Selectivity The fact that pervaporation can only be used to separate volatile analytes may initially seem a shortcoming rather than an advantage; however, it results in increased selectivity for the target analytes and enables their determination in complex samples such as biological fluids and environmental specimens [170] with no prior treatment, using pervaporation as a clean-up step. If the target analyte is not volatile but can be converted into a volatile species via a selective (bio)chemical reaction, it can also be separated by pervaporation. The analyte is made volatile by adding an appropriate reagent to the sample, either in a continuous manner or by direct injection into the matrix. In any case, a derivatization reaction is often required to form the species to be eventually detected. The possibility of employing the derivatization reaction prior to or after the separation step has extended the scope of pervaporation to a variety of analytes and allowed

142


pervaporation units to be coupled to various detector types. Pervaporation can also be used for speciation as it allows the selective determination of different forms of the target analyte. In multideterminations, and also when the target volatile analytes possess different boiling points, the pervaporation unit can be used at different temperatures to ensure selective separation of the species of interest. Use of a heating system allowing reproducible control of time and temperature is obviously mandatory here. Discrimination between polar and non-polar volatile analytes can be accomplished by using microwave irradiation of the donor chamber in order to facilitate the evaporation of polar species. Non-polar ones can then be evaporated by conventional heating (whether electrical or by use of a water bath). Selectivity can also be boosted by changing the membrane used in the separation process. As noted earlier, the permeability of glassy polymers is usually lower than that of rubbery polymers, while selectivity is primarily dictated by the molecular dimensions of the permeated species [167]. Precision

The fact that placement or passage of the sample in or through the donor chamber, respectively, is made quite reproducible by the use of stirring or chemically inert beads results in highly reproducible signals following pervaporation. Experiments conducted in the presence and absence of stirring and chemically inert beads revealed that significantly improved precision, as relative standard deviation, was obtained from the use of glass beads (0.79%) and magnetic stirring (1.78%) in relation to the absence of both (11.29%) [159]. Repeatability can also be improved by aspirating the detector waste at a flow-rate identical with that of the acceptor stream or, if a chemical reaction with an additional reagent is required, to the combined individual flow-rates [149]. When the concentrations of the target analytes are relatively low, a low precision is obtained that can be ascribed to the fact that equilibrium conditions in the gas phase held in the donor chamber are not readily reached. This shortcoming can be circumvented by previously saturating the air gap with the volatile species (i.e. by injecting the required volume of a standard solution containing an appropriate volatile concentration and driving it to the lower part of the separation module). The flow of both the donor and the acceptor stream is then stopped for a preset time and, once resumed, the experiment can be continued with fairly good results (the precision thus obtained in the determination of sulphide was 5.46% as RSD) [145]. The use of long waste tubes in the lower part of the pervaporation module can also improve the precision of the ensuing method since the pressure in the module is thus increased and small variations in pressure caused by both internal and external noise yield a smaller overall effect when the length of the waste tubing changes from 20 to 280 cm [147]. In this way, the RSD decreased from 5.46% to 2.85% for injection of the sample, and from 7.03% to 1.61% for continuous introduction of sample. Halting the flow of both the acceptor and the donor stream resulted in a repeatability as RSD of 2.51% at the normal waste tube length (20 cm) and 1.21% for longer lengths (280 cm) [147].


143

The use of gas-diffusion membranes instead of ultrafiltration membranes can also improve the precision as the former are not easily bent; this avoids changes in permeate flux resulting from both irreproducible membrane areas and increased volumes of the upper chamber [ 15 3,171 ].

4.4.8. Scope of application of analytical pervaporation Because the full potential of pervaporation remains to be realized, its applications are still small enough in number to be easily summarized. Table 4.7 shows such applications classified according to whether the sample was liquid or solid. Within each category, applications are classified according to field: environmental, food and clinical. A theoretical application to the calculation of Henry constants is illustrated at the end. The applications of pervaporation can also be classified according to whether they involve any derivatization reaction. Such reactions can be used prior to the separation step in order to convert the analyte into a volatile compound or subsequent to the mass transfer step (in the upper manifold) in order to obtain a product that can be measured by the detector. The reaction in question can be of chemical or biochemical nature. One other possible classification of pervaporation applications is according to whether a single or many parameters are determined. The feasibility of multideterminations relies on the separation process (viz. on the use of different temperatures for analytes with different vapour pressures), a reaction preceding or following pervaporation, the use of a discriminating separation technique or the characteristics of the detector. Whether the separation step is integrated with detection also gives rise to two types of methods. One of the most salient applications of pervaporation is no doubt its use as an alternative to both headspace modes (static and dynamic or purge and trap), which it surpasses in some respects. Below are briefly described various aspects of pervaporation applications. Sample types Although this discussion focuses on solid samples, pervaporation is applicable to liquids, solids and even slurries. As stated in Section 4.4.3, both the manifold used and the way the sample is inserted vary depending on whether it is liquid or solid. Also, as noted in the same section, slight alterations to the manifold are required to process slurries. The most salient difference between the treatment of liquid and solid samples is that, with the former, the whole process from injection of the sample to delivery of the results can be fully automated. By contrast, some human intervention is required in sample weighing and tight fastening of the pervaporation module for solid samples. Only partial automation is possible in this case as the rest of the process (viz. derivatization if required - - pervaporation, transport to the detector, monitoring, data acquisition and processing, and delivery of the results) can be performed in an automated fashion. Derivatization reactions When a derivatization reaction is developed in either the lower manifold (to form a volatile product) or the upper one (to adapt the monitored product to the characteristics

TABLE 4.7 GENERAL APPLICATIONS O F ANALYTICAL PERVAPORATION

Analyte

Sample type

Derivatization reaction

Detection

Special features

Ref.

Solid samples

F-

Tree leaves, fertilizers, ceramic industry wastewater

Chemical

Integrated potentiometry

Continuous-discrete manifold

165

Dichlorvos

Natural water. soil

Biochemical

Fluorimetry

Enzymic derivatization by inhibition

177

Metrifonate

Natural water. soil

Biochemical

Fluorimetry

Triple integration: chemical hydrolysis-pervaporationenzymic derivatizdtion by inhibition

178

AmmonidUrea

Soil

Biochemical

Integrated potentiometry

Sequential determination. Speciation analysis

I73

Mercury compounds

Soil (CRM)

Atomic fluorimetry

Microwave-assisted pervaporat ion. Speciation analysis

28

Mercury

CRM

Atomic fluorimetry

Alternative to headspace, speciation

I69

vocs

Soil

ECD'**'

Alternative to headspace

171

Acetaldehyde

Fruit juice, yoghurt, bread

Chemical

Photometry

Food samples

I75

Trimethy lamine

Fish

Chemical

Photometry

Pretreatment integrated with pervaporation

176

Liquid samples F-

Fertilizers, ceramic industry wastewater

Chemical

Potentiometry

Stopped-flow preconcentration

I49

s2-.

Kraft liquors

Chemical

Photometry

Improved precision and sensitivity

145,147

Cyanide

Samples from the mining industry

Chemical

Amperometry

Determination of cyanide in the presence of sulphide. Interference study

162

Phenol

Water

Chemical

Amperometry

Stopped-flow preconcentration

185

Ammonia

Industrial effluents

Chemical

Photometry

Determination in the presence of surfactants

191

COD'*)/lnorganic carbon

Bleaching liquors, domestic sewage, river water

Chemical

Photometry/ integrated potentiometry

Simultaneous determination of COD'*' and inorganic carbon

I66

Ethanol

Wine

Biochemical

Fluorimetry

Immobilized enzyme on-line monitoring fermentation

174

Ethanol

Fermentation media

Chemical

Photometry

On-line monitoring

193

Ethanol

Wine

Biochemical

Photometry

Immobilized enzyme

188

Acidity

Wine

Chemical

Photometry

Sequential determination of total and volatile acidity

183

Acidity

Wine

Chemical

Photometry

Determination of volatile acidity

186

so,

Wine

Chemical

Photometry

Speciation of free and bound SO,

150

co,/so,

Wine

Chemical

Photometry/ potentiometry

Simultaneous determination of c o , / s o 2

182

TABLE 4.7 (cont.) Analyte

Sample type

Derivatization reaction

Detection

Special features

Ref.

Chemical/ biochemical

Photometry/ fluorimetry

Simultaneous determination of ethanoliglycerol, immobilized enzyme Alternative to headspace Immobilized enzyme Electronically controlled temperature, immobilized enzyme Stopped-flow preconcentration During fermentation and in final product Immobilized enzyme, continuous monitoring Sequential determination, immobilized enzymes Continuous4iscrete manifold, immobilized enzyme Stopped-flow preconcentration Continuous-discrete manifold Theoretical studies, Henry constants

184

Liquid samples (cont.)

EthanoVglycerol

Wine

Volatile species Ethanol/diacet y l Ethanol

Wine Yeast extract Bakers' yeast culture, beer

Biochemical Biochemical

Fluorimetry Voltammetry, fluorimetry

Diacetyl

Beer

Chemical

Photometry

Diacetyl

Beer

Chemical

Adsorptive stripping

Acetaldehyde

Fermentation media

Biochemical

Photometry

AmmonidUrea

Serum, urine

Biochemical

Oxalate

Urine, serum

Biochemical

Conventional potentiometry Conventional potentiometry

Formaldehyde

Pharmaceuticals and cosmetics Pharmaceuticals and cosmetics Water

Chemical

F Phenols (*)

FIDI *** 1

Chemical

Conventional photometry Integrated potentiometry Amperometry

Chemical oxygen demand ( * * ) Electron capture detector ( * * * ) Flame ionization detector

I87 188 188

195 192 175

150 151

189 190 194


147

of the detector), the basic dynamic manifold assembly may have to be altered depending on the particular reaction to be used. Actions on the lower chamber intended to obtain volatile species involve injecting appropriate reagents for solid samples or merging a stream thereof with the channel that contains the injection valve at a point in between the valve and the pervaporator. When post-pervaporation reactions are needed, the most effective way of implementing them is by using the solution containing the reagents as acceptor solution. In this way, the pervaporated analytes react on the acceptor side of the membrane, thus displacing the mass transfer equilibrium. Solid reagents, which represent the most usual situation in enzyme-catalysed reactions, are packed in a solid reactor (a tube) placed between the injection valve and the separation module when the analyte in the liquid sample is non-volatile in order to obtain a volatile product amenable to separation; however, the solid biocatalyst-support conjugate can also be placed in the donor chamber for two main purposes, namely: (a) to increase the yield of the biocatalysed reaction (because the product is removed from the solution into the air gap and then into the acceptor solution, the reaction equilibrium is gradually displaced to quantitativeness); and (b) to avoid dispersion of the sample in the carrier solution when the analyte concentration is low and the sample scant (and hence inadequate for insertion in a continuous manner) [152]. When the sample is a solid, the use of the biocatalyst bonded to a solid support in the lower chamber is unfeasible unless the support consists of magnetic particles [ 172], which can be removed and reused after the analysis has been completed. Thus, enzymes are usually employed in dissolved form when a volatile product is to be obtained from a solid sample [173]. When the purpose of the biocatalysed reaction is to obtain a product amenable to detection, the reactor, packed with the support--enzyme conjugate, is placed between the acceptor chamber and the detector. The enzyme has also occasionally been immobilized in the upper chamber of the pervaporation module in order to integrate separation and an enzymatic reaction with an immobilized biocatalyst. This approach has been used to monitor ethanol production during wine fermentation and found to result in longer durability of the immobilized biocatalyst [174]. Individual determinations

The simplest use of pervaporation is for the separation and subsequent determination of a single analyte; as a result, most reported methods using this technique involve individual determinations. The determination of acetaldehyde in semi-solid and solid food samples is one salient example of single-analyte determinations where pervaporation avoids time-consuming sample preparation steps such as filtration, removal of dyestuffs and turbidity from the Carrez solution, and centrifugation [175]. One other example is the determination of trimethylamine (an objective parameter for fish quality evaluation that correlates well with sensory evaluation) [176]. The method is based on pervaporation of the analyte and monitoring of the colour change in Bromothymol Blue caused by the basic character of the amine; the results are consistent with those provided by the official method for trimethylamine. One typical example of the high potential of pervaporation for boosting the selectivity of processes such as enzymic inhibition-based reactions is the method for the selective determination of pesticides in environmental samples. The method, which is only

148


applicable to volatile pesticides or their volatile degradation products, has been used in the determination of diclorvos (boiling point 35~ [177] and metrifonate (a compound yielding diclorvos as hydrolysis product) [178]. Diclorvos, both directly and after hydrolysis, has been determined in soil following pervaporation by its inhibitory action on acetylcholinesterase oxidase catalysis. The decrease in the fluorimetric signal obtained after a two-step derivatization reaction involving choline oxidase and horseradish peroxidase is proportional to the inhibition caused by the analyte and hence to its concentration in the sample. The determination of pectinesterase activity in fruit and vegetables by monitoring the methanol released and pervaporated [179] is one other example of the potential of pervapotation for individual determinations in solid samples.

Multideterm inations Multideterminations involving pervaporation [180] can be implemented either by altering parts or parameters affecting the overall approach or by coupling the dynamic manifold of the upper chamber to a highly discriminating separation technique such as gas chromatography.

Multideterminations involving solid samples Determinations of this type using pervaporation have so far focused on speciation analysis [181]. The two most salient examples reported to date are the speciation of inorganic and organic forms of mercury (Fig. 4.23A) and that of nitrogen as ammonia and urea (Fig. 4.23B). The speciation analysis of mercury in soil was based on microwave-assisted pervaporation/atomic fluorescence detection in the straightforward, discrete-continuous manifold depicted in Fig. 4.23A. Focused microwaves proved highly efficient when the analytes were polar enough to interact with this type of energy; such was the case of HgNO 3 and PhHgOAc, for example. In the manifold used, the sample was weighed in the donor chamber of the pervaporation cell and the cell was tightly closed and connected to the loop of the injection valve accommodating the pervaporator's upper chamber. This allowed the carrier stream (water or argon) to pass either through the acceptor chamber and then into the detector, or via a bypass to the detector while the flow in the acceptor chamber was stopped. Although the pervaporator flow was stopped, the baseline did not change because the carrier stream proceeded to the gas-liquid separator via the bypass loop at the same rate as it was circulated through the pervaporator. Once the sample was in the donor chamber, the reagents were injected via the donor chamber inlet. Septa similar to those used in GC allowed the syringe needle to penetrate the donor chamber, inject the reagents and be removed with no loss of either sample or reagents. One minute after the reagent (acidic SnCI2) was injected, the microwave device was switched on at a preset power for a given time, after which the cell was allowed to cool for 1 min before the valve was actuated to divert the carrier through the acceptor chamber to collect one of the mercury compounds to the gas-liquid separator if the acceptor carrier was liquid or directly to the atomic fluorescence detector if it was gaseous. Once the detector baseline was restored, the valve was switched again to isolate the pervaporator, the reagents (viz. KBr/KBrO 3 as oxidant and SnCI 2 as reductant) were sequentially injected and the


149

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Fig. 4.23. Manifolds used for speciation analysis of solid samples 9(A) Mercury speciation (as Hg2' and PhHgH). (B) Nitrogen speciation (as ammonia and urea). GLS gas-liquid separator, MWS microwave system, ISE ammonia-selective electrode, RE reference electrode, WB water bath thermostat. Other abbreviations as in previous figures. (Reproduced with permission of Elsevier.)

microwave digester was switched on again for another preset time at an appropriate power setting for the other mercury - - now organic - - compound. After the peak was recorded, the valve was actuated to isolate the cell and the system was thus made ready for weighing the next sample [28]. The method was validated by using a certified reference material (viz. CRM 145R from the BCR). The speciation analysis of nitrogen (as ammonia and urea) in soils was based on an enzymatic reaction and integrated pervaporation/potentiometric detection using the manifold of Fig. 4.23B. The procedure also involved weighing the sample in the donor chamber, shutting the pervaporator and propelling the acceptor stream through the upper chamber until a steady baseline was obtained 9 Then, an appropriate volume of NaOH solution was injected into the donor chamber through the septum, the flow in the upper chamber being stopped and the signal provided by the selective electrode used (corresponding to the ammonia present in the sample) recorded until the steady state value was reached. The flow was then restored and the sample neutralized by injecting HC1, followed by Tris buffer at pH 8.8 and an appropriate volume of urease solution in the same buffer.

150


The flow was stopped again and the signal corresponding to the urea concentration in the sample was recorded as before. During measurements, the pervaporation module was kept in a thermostatted water bath at the required temperature [173]. Multideterminations involving liquid samples These are easier to implement and hence more common than the previous ones. Some involve speciation while others have a different analytical purpose. Pervaporation-based speciation analyses of liquid samples have provided methods for determining two volatile species or one volatile species (determined after passing into the acceptor chamber) and a non-volatile one (determined either in the effluent from the donor chamber or by using another sample aliquot injected simultaneously with that driven to the pervaporator). The method for the determination of free and bound SO 2 in wine [150], and that for ammonia and urea in serum and urine [ 151 ], are typical examples of sequential speciation analyses of two volatile species. On the other hand, the methods for measuring chemical oxygen demand and inorganic carbon in bleaching liquors and domestic sewage [166] are examples of volatile and non-volatile speciation analyses. Monitoring wine fermentation and determining the final product pose special challenges as they involve analyses of liquid samples containing a number of volatile compounds and a matrix that is complex enough to require the use of pervaporation. Typical examples testifying to the high potential of pervaporation in this context include a method for determining two volatile species by using either a single photometric detector (for free and bound SO2) [150] or two serially arranged detectors (a potentiometric one for CO 2 and a photometric one for SO2) [182]; one for the determination of a volatile species and a non-volatile one with a single photometric detector (for volatile and total acidity) [183]; and one employing a photometric detector and a fluorimetric one arranged in parallel for ethanol and glycerol, respectively [184]. Coupling a pervaporator to a gas chromatograph. an alternative to headspace sampling

The use of pervaporation as an alternative to the headspace technique is worth separate discussion. This is, in fact, one of the most promising uses of this approach, as revealed by two existing methods for mercury speciation and VOC analysis in solid samples that exemplify the advantages of pervaporation over static and dynamic headspace modes. Both methods were developed by using the overall assembly depicted in Fig. 4.24A, by which the analytical process was developed in the following four steps: (1) Pervaporation of the analytes. An amount of ca. 0.5 g of sample was placed in the pervaporator's donor chamber, which was then closed, connected to the system and placed either in a water bath or in a vessel of a focused microwave device depending on the polar or non-polar nature of the target analytes. These were evaporated into the gas layer above the sample and then diffused through the membrane to the argon acceptor stream. (2) Preconcentration/desorption. A Tenax minicolumn accommodated in the loop of an injection valve which was placed in an ice bath was used to preconcentrate


151

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Fig. 4.24. Coupling a pervaporator to a gas chromatograph for (A) solid and (B) liquid samples. I chromatograph injector, GC gas chromatograph, FID flame ionization detector, PC personal computer. 1, 2 and 3 denote pervaporation, retention and chromatographic steps, respectively. Other abbreviations as in previous figures. (Reproduced with permission of Elsevier and Springer-Verlag, respectively.)

152


the volatile species passing through the membrane in the pervaporation unit. In order to desorb the retained species and inject them onto the chromatographic column, the flow of argon from the pervaporation unit was diverted so that the valve loop could be shut off with no gas flow passing through it. The Tenax minicolumn was removed from the ice bath and placed in a muffle oven at an appropriate temperature for nonpolar analytes or in a microwave device for polar analytes, following which the valve was switched to the inject position to allow a helium stream to pass through the column and flush the desorbed species from the loop of the injection valve through the connector between the injection valve and the chromatograph, and onto the chromatographic column. (3) Chromatographic separation. The helium stream containing the desorbed compounds entered the injector in a continuous manner via a hypodermic needle fitted to the injector inlet. This stream was then merged with an additional one flowing through the normal carrier gas inlet into the injector. The chromatograph oven was programmed as required and the species separated. (4) Detection of the isolated species. As the individual mercury species emerged from the chromatographic column, they were passed through a pyrolysis unit at 800~ in order to break the compounds down to elemental mercury. The resulting stream was then mixed with a make-up gas on its way to the detector. A second stream of the same gas (argon) was used as sheath gas, which increased the reproducibility as a result of the He/Ar/Hg flow being maintained in the beam of the atomic fluorescence detector. VOCs required no pyrolysis unit, so the isolated analytes were driven directly to the electron capture detector (ECD). No hydrophilic membranes could be used as they allowed the passage of water, which then reached the GC column. PTFE membranes were found to provide increased sensitivity and thermal stability relative to other hydrophobic membranes such as those of the PVDF type [171]. The coupling of an analytical pervaporator to a gas chromatograph for the determination of enological parameters in wines is an excellent example of the usefulness of this approach for the analysis of liquid samples. The Fl-pervaporation-GC assembly of Fig. 4.24B differs from that used with solid samples in the need to insert the sample by injection and the fact that no preconcentration/desorption step is required. The process involves the following steps: (1) Pervaporation/preconcentration of the analytes. First, the loop of the injection valve (IVI in Fig. 4.24B) is filled with sample while IV2 and IV3 are in their injection and filling positions, respectively. In this way, the donor stream (H20) is continuously circulated through the lower chamber of the pervaporation unit and the helium stream is continuously sent to the gas chromatograph; meanwhile, a portion of the helium remains static in the acceptor chamber to collect the volatile species. Then, IV1 is switched to the injection position and the sample is transported by the H20 stream through the lower chamber of the pervaporation unit, which is immersed in a thermostatic bath at 80~ (2) Injection, chromatographic separation and detection. Five minutes after the sample is injected this is the optimum preconcentration time IV2 is switched to its


153

filling position and, immediately, IV3 to its injection position for 8 s. During this interval, the evaporated species are carried out to the top of the chromatographic column and, after separation, driven directly to the flame ionization detector. The upper chamber of the pervaporation unit requires flushing before a fresh sample is injected. To this end, IV2 and IV3 are switched to the filling and injection position, respectively, for 10 s. Pervaporation provides a number of advantages over the headspace technique that can be summarized as follows: (a) The thin air gap layer above the sample requires very small amounts of the analytes to establish equilibrium with the solid or liquid sample phase and mass transfer across the membrane. (b) Continuous removal of the volatilized analytes across the membrane from the air gap displaces the mass transfer equilibrium and increases the separation efficiency. (c) Continuous removal of the pervaporated analytes to the preconcentration column can be used to allow fresh portions of acceptor gas to continuously come into contact with the diffused species, thus displacing the mass transfer equilibrium. (d) The separation process can be easily automated for laboratory use with minimal purchase and maintenance costs. (e) Unlike purge and trap, no water vapour condenser is required, nor is a hydrophobic sorbent as no water-crossing through the (hydrophobic) membrane occurs. Integrated pervaporation and detection The best way of monitoring pervaporation kinetics is by inserting a probe-type sensor in the acceptor chamber, with its active side facing the membrane. A number of methods based on this principle have been reported. The determination of fluoride in liquid (ceramic industry wastewater, dissolved fertilizers) and solid samples (e.g. tree leaves) by formation of a volatile product with hexamethyldisiloxane using a manifold such as that of Fig. 4.23B is an excellent example of this approach [165]. The most salient advantages of integrating pervaporation and detection are as follows: (a) the response time is shortened with respect to the conventional location of the sensor behind the separation module as the need to transport the target species from the module to the detector is avoided; (b) the kinetics of mass transfer across the membrane can be monitored in order to obtain a better understanding of the separation step and easier, in-depth optimization; and (c) the size of the set-up required for the determination of a volatile analyte or reaction product in solid samples can be dramatically reduced by integrating the leaching, derivatization, separation and detection steps. Table 4.7 includes additional analytical uses of pervaporation [185-194]. Detector types Pervaporators are amenable to coupling to any type of detector, whether molecular or atomic, via an appropriate interface such as a transport tube, a microcolumn packed with

154


adsorptive or ion-exchange material, or a gas-liquid separator. The acceptor carrier stream can be either liquid or gaseous depending on the characteristics of the detector. The detectors most frequently used in conjunction with pervaporation are of the spectroscopic [atomic (fluorimetric, absorptiometric) or molecular (photometric, fluorimetric)], electroanalytical (potentiometric, voltammetric), electron capture and flame ionization types. The low selectivity of some detection techniques such as photometry, ion-selective electrode-based potentiometry and flame ionization is offset by that of the pervaporation step, which endows the overall analytical process with the selectivity required to process highly complex matrices. The potential of the pervaporation technique for sample insertion into water-unfriendly detectors such as mass spectrometers or devices such as those based on microwave-induced plasma remains an unexplored field for pervaporation.

4.4.9. Prospects for analytical pervaporation As shown above, pervaporation is a useful analytical tool that features simplicity, and automation and miniaturization capabilities [195-197]. The actual potential of this technique for the pretreatment of solid, liquid and slurry samples can readily be inferred from its intrinsic features and from available methods using a pervaporator. Consolidation of this technique as an effective, widespread analytical tool is bound to rely on future, sequential developments such as the following: (a) the gathering of sufficient research experience to compile a cookbook of methods where potential users can search for solutions to specific problems; and (b) the commercialization of inexpensive pervaporation modules meeting the requirements of a variety of samples and analytes. The development of such methods and theoretical studies should turn pervaporation into a useful tool for routine environmental, clinical, food and industrial analyses.

4.5. SOLID-PHASE MICROEXTRACTION

4.5.1. Introduction The idea of using a sorbent material to extract trace organic compounds from, in principle, an aqueous sample, emerged as sorbent extraction was developed a few years ago [198]. At present, sorbents are used to extract organics from matrices such as water, air and soil. One of the most salient features of this technique is that it allows analytes to be concentrated by the sorbent. In fact, by using a sorbent with a strong affinity for the target analytes, these can be concentrated from an otherwise highly dilute sample. Solid-phase extraction (SPE) is one of the most commonly used sorbent extraction techniques. The analytes are extracted together with interfering compounds with some similar characteristics by passing a usually aqueous sample through a plastic cartridge containing dispersed sorbent on a particulate support. A selective organic solvent is normally used to remove interferences and a different solvent is then employed to elute the retained analytes. This technique has a number of attractive features as compared with traditional solvent extraction. Thus, it is quite simple and inexpensive, can be automated and used in the field, and uses relatively small amounts of solvents. Particle-load

Solid sample treatments invoh,ing the removal of volatile species

155

membranes known as Empore extraction discs have improved extraction efficiency, decreased solvent consumption and reduced plugging in SPE since their introduction in 1990 [199]. However, SPE still has some limitations such as the low recoveries that result from interactions between the sample matrix and analytes, and plugging of the cartridge or blocking of the pores in the sorbent by solid or oily components, which results in low breakthrough volumes and also low capacities. Although Empore discs have to some extent overcome these drawbacks [200], some problems still exist, along with high blank values and batch-to-batch changes in the sorbents. Because SPE is a multi-step approach involving concentration, it is restricted to semi-volatile compounds with boiling points substantially above those for the solvents. One solution to the shortcomings of SPE is to improve the sorbent geometry by coating it on a fine rod such as a fused-silica fibre or a wire of an appropriate material. The cylindrical geometry of the resulting system ensures rapid mass transfer during extraction and desorption, prevents plugging, and facilitates handling and insertion into analytical instruments [201]. This approach is known as "solid-phase microextraction" (SPME) [202-204]. Because SPME is a static extraction technique, the need for a large surface area is no longer as critical as in SPE. Smooth liquid coatings can be used that avoid plugging. Also, by sampling from headspace, SPME can extract analytes from highly complex matrices such as sludge. 4.5.2. Principles, devices and theoretical aspects of SPME Principles and devices

A solid-phase microextraction process involves two steps, namely: partitioning of the analytes between the coating and the sample, and desorption of the concentrated species into an analytical instrument. In the first step, the coated fibre is exposed and the target analytes are extracted from the sample matrix into the coating. In the second step, the fibre with the concentrated analytes is transferred to an instrument for desorption. A third, clean-up step can also be incorporated by using selective solvents, as in SPE. SPME applications have focused on the extraction of organics from various matrices including air, water and soils, followed by direct transfer of the retained species into a GC injector, desorption from the injector, separation on the chromatographic column and measurement by the detector. A fused-silica fibre coated with a gas chromatographic stationary phase such as polydimethylsiloxane is typically used for the microextraction. This type of fibre and its coating are obtained by using well-established techniques for manufacturing optical fibres. The fused-silica fibre itself is chemically inert and very stable, even at high temperatures. The small size and cylindrical geometry of the fibre allow it to be incorporated into a syringe-like device such as that shown in Fig. 4.25, which can be operated like an ordinary syringe and easily accommodated in a GC injector. As shown in the figure, the fused-silica fibre is connected to stainless steel tubing that is used to increase the mechanical strength of the fibre assembly for repeated sampling. The stainless steel tube is contained in a specially designed syringe. During SPME, the fibre is first withdrawn into the syringe needle and then lowered into the vial by pressing

156


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Fig. 4.25. SPME device and detail of the fibre and needle. (Reproduced with permission of the American Chemical Society.)

down the plunger. The fibre coating is exposed for a preset time to extract the analytes from their matrix. Once the sampling is completed, the fibre is directly transferred into a gas chromatographic injector. The analytes are thermally desorbed from the fibre coating and quantitatively analysed by GC. The steps involved in an SPME process can be developed either manually or automatically (with the aid of an autosampler). Because the SPME device is basically a syringe, anyone who can use a syringe properly can implement this extraction technique. The main difference between a typical autosampler and those used in SPME is that the plunger movement and timing must be carefully controlled

Solid sample treatments involving the removal o[volatile .species

157

to ensure correct adsorption and desorption. When SPME is used for sampling in the field and subsequent analysis in the laboratory, special care should be exercised to avoid analyte losses during transport. The needle opening of the SPME device can be sealed with a piece of septum and/or by cooling the needle. Because increased temperatures have an adverse effect on adsorption, the thermal desorption of analytes from an SPME coating is highly effective in most cases. As the temperature increases, coating/gas partition coefficients decrease and the ability of the coating to retain the analytes drops very rapidly. In addition, the constant flow of carrier gas within a GC injector facilitates the removal of analytes from the coating. For volatile and semi-volatile compounds, analytes can be desorbed from the coating at a temperature between 150 and 250~ in a fraction of a second and then focused at the front of the GC column [205]. The injection band can be further shortened by using an injector that can generate a heating pulse instead of constant heating; by using an internal heating device inside the fibre; or by directly passing the current through a fibre made of metal (a wire), as shown in Fig. 4.26. With compounds of a high molecular weight such as

E--lIE ] MODIFIED SYRINGE BATTERY CAPACITOR

INJECTOR NUT SEPTUM

CARRIER GAS INLET

METALLIC FIBRE

METAL J CONTACT

TO CAPILLARY COLUMN OR MASS SPECTROMETER

Fig. 4.26. Device for electrically heating an SPME fibre made from a metallic material. (Reproduced with permission of the American Chemical Society.)

158


perylene, however, carry-over may be a problem. No special thermal desorption module or alteration of the gas chromatograph is needed. The combination of SPME and thermal desorption completely eliminates organic solvents from extraction and injection, and integrates both processes into a single step. Desorption from the fibre can also be accomplished by using an appropriate solvent, a condensed medium such as compressed CO, or even an aqueous solution. For example, the selective extraction of bismuth from a fibre coated with an ion-exchange polymer can be accomplished by using an acidic potassium iodide solution as a complexing reagent [206]. The most important analytical parameters (viz. throughput, sensitivity, accuracy and precision) of SPME are largely determined during the extraction step. Desorption, which strongly influences the efficiency of the chromatographic separation and the precision of the quantitation, largely dictates the quality of the results and the scope of SPME. Theoretical aspects

The principle behind SPME is the partitioning of the analytes between the sample matrix and the extraction medium. When a liquid polymeric coating is used, the amount of analyte absorbed by the coating at equilibrium is directly related to its concentration in the sample: m=

K,~V,C,,V, K,~Vt + V~

(4.45)

where m is the mass of analyte adsorbed by the coating; Vt. and V~ are the volumes of the coating and sample, respectively; Kta is the partition coefficient of the analyte between the coating and the sample matrix; and C 0 is the initial concentration of the analyte in the sample. Equation (4.45) establishes a clear-cut linear relationship between the amount of analytes adsorbed by the fibre coating and their initial concentration in the sample. Because the coatings used in SPME have strong affinities for organic compounds, the Kt~ values for the target analytes are quite large; this means that SPME exerts a very strong concentrating effect and hence results in good sensitivity. In many cases, however, Kta values are not large enough to ensure thorough extraction of most analytes in the matrix. Rather, SPME, like the static headspace mode, is an equilibrium sampling method and, through proper calibration, can be used to accurately determine the concentrations of target analytes in a sample matrix. As can be inferred from eq. (4.45), if V, is very large (i.e. if v, >> Kf:~VO, the amount of analyte extracted by the fibre coating, m = K,~Vt.C o

(4.46)

is not related to the sample volume. This feature, combined with its straightforward geometry, makes SPME suitable for field sampling. Because the fibre can be exposed to air or dipped directly into a well, lake or river, for example, SPME reduces field analysis time by combining sampling, extraction, concentration and injection into a single, uninterrupted process.


159

The speed of extraction is controlled by the mass transport of the analytes from the sample matrix to the coating. This process involves convective transport in an air or liquid sample, desorption of the analytes from the solid surface when particulate matter is present, and diffusion of the analytes in the coating [205]. In direct SPME sampling, the mass transfer rate is determined by the diffusion of analytes in the coating provided the sample matrix is thoroughly agitated. When the rate of mass transfer is determined by the diffusion of the analytes in the coating, equilibrium is reached within less than 1 rain for most analytes [205]. The small thickness of the coating (typically between 10 and 100 ~tm) ensures rapid extraction. In practice, this limit can only be achieved for gaseous samples, which possess large diffusion coefficients. With aqueous samples, equilibration requires the use of vigorous agitation methods such as sonication [207]. With more common agitation methods such as magnetic stirring, the equilibration time is much longer and determined by diffusion through a thin static aqueous layer adjacent to the fibre. Such a thin layer of water around the fibre is very difficult to remove even when water is rapidly stirred to enhance the mass transfer of analytes; the analytes must diffuse through the water before they can be adsorbed by the fibre coating. Placing a fibre directly into a sample to extract organic compounds works well with gaseous samples and relatively clean water samples. Sampling analytes from a solid matrix of wastewater containing grease, oil or high-molecular weight humic acids is not so easy with SPME, however, and requires using the headspace above the sample matrix. The SPME technique can thus be used to extract organics from virtually any type of matrix provided the target compounds can be released from the matrix into the headspace [208]. For volatile compounds, releasing analytes into the headspace is relatively easy because they tend to vaporize once they are dissociated from the matrix. For semi-volatile compounds, the low volatility and relatively large molecular size may slow down the mass transfer from the matrix to the headspace and, in some cases, the kinetically controlled desorption or swelling process can also slow down the extraction. When the matrix absorbs the analytes more strongly than the extracting medium does, the analytes partition poorly into the extraction phase. Because of the limited amount of the extraction phase in SPME, the extraction will be subject to a thermodynamic constraint: the partition coefficient, Kf~, will be too small and result in poor sensitivity. If the coating has a stronger ability to adsorb the analytes than the matrix does, it will only be a matter of time for a substantial amount of the analytes to be extracted by the fibre coating and only kinetics will play a significant role during extraction. One of the most efficient ways of overcoming the kinetic constraints is by heating the sample, which increases the vapour pressure of the analytes, provides the energy required for analytes to dissociate from the matrix and results in more expeditious transport of the analytes. Headspace SPME involves three phases (viz. coating, headspace and matrix), the chemical potential difference of the analytes between the phases being the driving force that moves them from the matrix to the fibre coating. With aqueous samples, headspace/water partition coefficients (Kh,) are directly related to the Henry constants for the analytes, which are in turn determined by their volatility and hydrophobicity.

160

Solid sample treatments involving the removal of volatile species 30 000 000

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480

TIME, s

Fig. 4.27. Adsorption-time profile for 0.75 lag/ml trichloroethene in both stirred and unstirred solutions. (Reproduced with permission of the American Chemical Society.) In aqueous matrices, most compounds have quite small Kh~ values (< 0.25), so the headspace has a low analyte trapping capacity. As a result, the sensitivity of headspace SPME is almost the same as that of direct SPME. The sensitivity loss is only significant when the target analytes partition well into the headspace (i.e. when they possess large Kh., values) and/or a large headspace volume is used. For aqueous VOCs, headspace SPME sampling is faster than direct SPME. In headspace SPME, mass transfer from water to the headspace can be rapidly expedited by continuously stirring the water sample to generate a continuously renewed surface (see Fig. 4.27). The mass transfer of volatile compounds from the headspace to the fibre coating is very fast because of the large diffusion coefficients of the analytes in the gas phase, and volatile compounds are more efficiently transferred from water to headspace to coating than from water directly to coating [208]. When the rate-determining mass transfer step is the mass transport of the analytes from the matrix to the headspace, the extraction time profile curve exhibits an initial steep rise (associated to partitioning of the analytes originally present in the gaseous headspace) followed by a portion with a gentler slope (resulting from slower mass transport of the analytes from the matrix). The rate of mass transfer is dictated by convective and diffusive transport of the analytes in the sample matrix [208] and/or by the slow desorption kinetics as in SFE [209]. Because partition coefficients are temperature-dependent, there is usually an optimum temperature for headspace SPME. As the temperature rises, more analytes are released from the matrix to the headspace, a process that results in high analyte concentrations in the headspace and favours extraction. At high temperatures, however, coating/headspace partition coefficients decrease and the matrix-dependent optimum extraction temperature


161

is determined by interactions among the analytes, coating and matrix. With an appropriate coating, headspace SPME can be used to extract both volatile and semi-volatile compounds from their matrices. Although SPME is mainly an equilibrium extraction technique, it has the ability to provide thorough extraction. If the coating/matrix partition coefficient, K~, is very large (i.e. KrsVf >> V), the amount of analyte desorbed by the coating will be m = C0K

(4.47)

and thorough extraction achieved. Coating types Because different groups of analytes can be extracted by different types of sorbents, SPME has been used with a variety of sorbents. For organics, the basic principle of"like dissolves like" applies. Polar coatings such as polyacrylate and Carbowax "- extract polar compounds such as phenols and carboxylic acids very efficiently, whereas non-polar coatings such as polydimethylsiloxane retain hydrocarbons very well. A gold coating works for extraction of mercury and an ion exchanger extracts metal ions. As shown in Table 4.8, SPME can be used to extract a wide range of compounds from various matrices by adjusting the operating conditions.

4.5.3. Variables affecting SPME The variables that influence SPME performance can be classified according to whether they relate to the retention step or the desorption step. TABLE 4.8 CHARACTERISTICS AND SCOPE OF SPME MODES Mode

Treatment

Analytes

Direct SPME

Routine In situ chemical derivatization In situ redox reaction

Most compounds Polar compounds

Routine

Volatile and semivolatile compounds Volatile and semivolatile compounds with small partition coefficients Polar compounds

Headspace SPME

Heating/cooling

In situ chemical derivatization

Matrices

Gaseous and liquid Inorganic ions

Any

162


Retention-related variables

Using an appropriate stationary phase is the key to ensuring efficient removal of the target analytes. Both the type of coating material and its thickness influence performance here. Stationary phases for SPME are commercially available in different thicknesses and polarities for a variety of analytes. As noted earlier, a polydimethylsiloxane phase is preferable for non-polar analytes and a polyacrylate phase for polar ones. For example, the extraction efficiency for triazine pesticides can be increased by a factor as high as 10-20 simply by using a polyacrylate phase rather than a polydimethylsiloxane one. Also, polydimethylsiloxane, which is a non-polar coating, extracts non-polar compounds such as BTX (benzene, toluene, ethylbenzene and xylene isomers) and PAHs (polycyclic aromatic hydrocarbons) from water very effectively but cannot extract polar compounds such as phenol or its derivatives (see Fig. 4.28A). On the other hand, polyacrylate, a more polar coating, extracts phenol and its derivatives quite well, but is relatively inefficient with BTX compounds (see Fig. 4.28B). The coating/water partition coefficients of BTX for polydimethylsiloxane are much greater than those for polyacrylate, whereas the opposite is true for phenol and its derivatives. For compounds with a large molecular size such as PAHs, large Kr, values can be obtained by using the polydimethylsiloxane coating. In fact, quantitative extraction of these analytes from aqueous matrices can be achieved because of their very large coating/water partition coefficients [210]. Two recently introduced phases (viz. Carbowax"-divinylbenzene for alcohols and divinylbenzene-polydimethylsiloxane for volatile amines) are more selective. The latter material is available in various thicknesses. A thick stationary phase is more suitable for volatile compounds, whereas a thinner phase is more efficient with larger molecules. Testing of various types of needles has revealed good manufacturer control of the stationary phase thickness that results in good reproducibility (3-9% as RSD) [211]. The repeatability of extraction with the same fibre depends on phase thickness and is better with thicker phases, but is adversely affected by stirring (particularly when extracting by hand). One recent development in this context is an HPLC stationary phase of 5 ~tm particle size glued to the metal needle. Silica, Cs and C~x reversed phases have been tested for this purpose. Adsorption of the analytes was found to be faster and fibre capacity to increase as a result of the larger active surface of the stationary phase. The mechanical stability of the needle was also increased as a result [212]. The extraction time is dependent on the partition coefficient of the analyte and on agitation of the sample, and is generally shorter for extractions from the headspace. With sonication, the equilibration time is close to 1 min [207]. With other agitation methods such as magnetic stirring, the equilibration time is typically 2-60 min, depending on the agitation rate and partition coefficient. Ensuring that the maximum possible amount of analyte is extracted entails reaching the equilibration time; this, however, is too long and impractical for many compounds. Usually, an absorption diagram (viz. an extraction-time profile curve such as that of Fig. 4.29) is constructed for the target analyte that shows the dependence of the amount of analyte absorbed on the extraction time. From such a curve, the shortest acceptable time as regards analyte detection is chosen. The sample temperature has a twofold effect when working with liquid samples: diffusion coefficients in water increase and extraction times decrease with increasing

163


A) o-xylene K=794

Iz iJJ n; n; 0 z 0

2,4-d ich Ioro phenol K=4.6

.,--I

0 F-

)L

I

I

15:00

10:00

B) I-z i.u n,' n,' E~ ~O z

O

2,4-dichlorophenol K=47

._J

< IO I-

,, ^J

o-xylene K=5.4 fl

iA

I

15:00

10:00 TIME, min

Fig. 4.28. Total ion current GC-MS chromatogram for BTX and phenol mixtures in water. (A) Extracted with a polydimethylsiloxane coating. (B) Extracted with a polyacrylate coating. K is the coating/water partition coefficient of the analyte, which drops by more than 2 orders of magnitude for o-xylene and increases by more than 1 for 2,4-dichlorophenol upon changing the coating from non-polar polydimethylsiloxane to polyacrylate. (Reproduced with permission of the American Chemical Society.)

164

Solid sample treatments involving the removal of volatile species 60

~

50-

_

.

=

.

_

_

=

_

~

-

-

=

.

=

=

-

40-

30-

20-

10-

I

5

I

10

I

15

20

ABS. TIME, min

Fig. 4.29. Extraction-time profile curves (signal height versus absorption time) for organochlorine pesticides in aqueous solutions. Conditions: spiking level 0.2-1.2 ng/ml; polydimethylsiloxane fibre length 100 ~tm; magnetic stirring rate 250 rpm. Although equilibrium was not reached for some analytes, the extraction time was chosen to be 15 min. (0) a-hexachlorocyclohexane, (11) fl-hexachlorocyclohexane, (A) dieldrin, (x) aldrin, (O) 4,4'-DDT, (O) 4,4'-DDE, (+) 4,4'-DDD and (D) endosulfan sulphate. (Reproduced with permission of Elsevier.)

temperatures, which, however, also result in decreasing partition coefficients. Heating the sample is therefore inadvisable except to expedite the release of analytes into the gaseous phase particularly for extraction from the headspace above solid samples. Improved transport of compounds from the sample to the gaseous phase can be achieved by heating the sample or by adding water or some other surface-active compound [208,213]. Microwave heating can also generate convective currents in samples that improve transfer of analytes from the matrix to the extracting phase. The sample pH is a crucial variable for slightly acidic or basic compounds. Extraction

Solid sample treatments involving the removal (?.]volatile species

165

TABLE 4.9 EFFECT OF THE ADDITION OF NEUTRAL SALTS ON SPME EFFICIENCY Peak area/lff', arbitrary units Compound

No salt added

Saturated with NaCI

a-BHC T-BHC Dieldrin Aldrin 4,4'-DDT 4,4'-DDE 4,4'-DDD Endosulfan sulphate

8.6 3.4 12.3 17.2 5.6 35.8 39.2 17.9

20.9 10.0 17.8 23.2 3.1 23.2 37.3 23.1

is more effective if such compounds are kept undissociated, as is well-known from liquid-liquid extraction and SPE procedures. The fibres should be handled with care as, for example, the polydimethylsiloxane phase is not resistant to media with pH below 4 or above 10 [214]. The ionic strength of the solution, when working with liquid samples, also affects the extraction step. Because the partition coefficients of the analytes (Kt~) are partly determined by the interaction between the target analytes and the matrix, the nature of the matrix can be altered to change the coating/matrix partition coefficients of the analytes. The salts commonly used for this purpose include NaC1 and Na2SO 4, which raise the ionic strength of water, thereby favouring the partitioning of polar organics but not ions into the polymer coating. As can be seen from Table 4.9, one potential drawback of using a salt to increase the ionic strength is a loss of fibre selectivity. The presence of organic solvents" in the sample suppresses the analyte absorption onto the fibre. Thus, Kt~ for benzene is decreased by 20% in the presence of 3% methanol [214]. Other well-sorbing hydrophobic substances such as dissolved organic carbon in natural water can have a similar effect. Air moisture also has some influence on the effectiveness of extraction from the headspace; in fact, at a relative humidity above 90%, absorption of analytes onto the fibre can be reduced by as much as 10% [215]. Derivatization can be used to reduce the polarity of polar compounds such as phenols and carboxylic acids, and also to increase their partition coefficients and improve the chromatographic separation. When working with liquid samples in SPME, polar analytes can be derivatized in their aqueous matrix (e.g. by converting phenols into their acetate derivatives) and then SPME applied [211]. With solid samples, derivatization is accomplished by doping the fibre coating with an appropriate reagent [216,217]; during the partition, polar analytes are simultaneously extracted and derivatized to less polar analogues that are amenable to GC analysis. Because the amount of reagent in the coating

166


is much higher than the equilibrium concentration of the acids, the reaction follows a pseudo first-order kinetics and the amount of derivative accumulated by the fibre is proportional to the integral of the acid concentration in air during the time of exposure. For example, the highly polar propionic and butanoic acids can be derivatized within the fibre coating into the pyrenyl ester, which has a high affinity for the coating and can be easily analysed by GC or HPLC. Problems such as low reaction recoveries or the formation of side products are to be expected in some cases, however. Also, the usual in-solution derivatization procedures are not always applicable to on-fibre derivatization because factors such as pH or the presence of certain catalysts are difficult to control.

Special aspects of extraction from solid samples Solid samples pose special problems in SPME as they cannot be extracted in a direct manner; rather, analytes must first be released into the headspace and then concentrated in the fibre coating. Some soil matrices such as sand exhibit relatively weak analytematrix interactions, which allows the ready headspace SPME sampling of volatile compounds. Others such as clay with a high metal content and large surface area exhibit strong analyte-matrix interactions and make headspace sampling difficult. With thermally stable analytes, heating the sample is an effective, convenient manner of releasing the analytes from the matrix to the headspace and improving sensitivity. A small amount of water and/or some other surface-active compound can also be added to soil samples in order to facilitate the release of analytes. For example, the amount of volatile hydrocarbon compounds extracted from soil matrices by SPME increases significantly when the sampling is conducted at high temperatures and a 10% moisture content [218]. Heating solid samples helps to release analytes from the headspace and facilitates extraction during SPME sampling. As the temperature increases, however, the fibre coating gradually loses its ability to absorb analytes. An optimum temperature therefore usually exists for SPME. If sampling can be done at a high temperature and a low coating temperature can be maintained at the same time, Kt~ and sensitivity are dramatically increased as a result. By altering the SPME device and using liquid CO_, as coolant, the sample can be heated to 250~ while maintaining the coating temperature below 20~ By using a high temperature, additional water, and cooled fibre, quantitative extraction of toluene, ethylbenzene and xylene i s o m e r s - which possess small partition coefficients and are impossible to extract quantitatively under room-temperature conditions can be achieved [210]. SPME from solid samples does not necessarily require the use of the headspace technique. Thus, aqueous suspensions of samples have proved effective for analytes not too strongly bonded to the matrix [219]. When the suspension is too thick, the fibre coating can be damaged during agitation. One alternative approach involves combining SPME with some other extraction technique such as superheated water extraction [220] or conventional liquid-liquid extraction (LLE) [221]. In the latter case, SPME is used as a fast alternative to time-consuming clean-up procedures and has proved especially effective for rapid screening. Quantitativeness in the determinations is hindered by strong analyte-matrix interactions, which can only be overcome by thorough extraction.


167

Desorption-related variables

The efficiency with which analytes are thermally desorbed from the SPME fibre is dictated by temperature, the desorption time and the position of the needle in the GC injector. The injectors in most modern gas chromatographs are amenable to direct insertion of the fibre, the only condition being that the liner for capillary GC should contain no packing. Although it has not been checked, desorption from the fibre and subsequent GC analysis are probably unfeasible with packed columns owing to the shape difference from the detector. The liner volume affects the shape of the chromatographic peak (larger volumes cause peak tailing). Split-splitless injectors should be operated in the splitless mode. The desorption temperature usually ranges from 150 to 250~ for semi-volatile compounds. Usually, the optimum desorption temperature is roughly equal to the boiling point of the least volatile analyte [214]. In order to prevent broadening of the chromato140

120 -

100

J

I

J

lJ

/x

-

v

E E

80-

i

-r

(.9

uJ T ._1 < Z (.9 00

60-

40-

20-

I

1

1

2 DESORPTION TIME, min

Fig. 4.30. Effect of desorption time on signal height. Sample spiked with chlorophenols and narrowbore liner. (0) 3-chlorophenol, (11) 2,5-dichlorophenol, (A) 2,3,5-trichlorophenol, (+) 2,3,4,6-tetrachlorophenoi. (Reproduced with permission of Elsevier.)


168

120

100 -

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(.9

60-

Z

40-

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x~~X~~x

O3

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0 0

I

I

2

3 FIBRE POSITION

Fig. 4.31. Effect of fibre position (graduation on fibre holder) in the GC injector (narrow-bore liner) during desorption. Sample: water spiked with chlorophenols. The reduction in signal height between the two extreme positions was less than 10%. (0) 3-chlorophenol, ( I ) 2,5-dichlorophenol, (/k) 2,3,5trichlorophenol, (x) 2,3,4,6-tetrachlorophenol. (Reproduced with permission of Elsevier.)

graphic peaks, the GC column should be kept at a low initial temperature or even cooled (cryofocused). An initial temperature 80-100~ below the boiling point of the most volatile analyte is recommended [214]. This ensures efficient concentration of the analytes at the column head. SPME and desorption can be repeated several times from the same sample and the analytes concentrated at the front of the cooled column. This, however, is rather time-consuming. The constant flow of carrier gas within a GC injector also facilitates the removal of analytes from the coating. The desorption time depends on the desorption temperature; in general, however, it is less than 1 s, so 1 minute's desorption in the injector should be more than adequate [214] even though considerably longer times are occasionally used (see Fig. 4.30). In any case, it is better to leave the fibre in the GC for a period of time (5-10 rain) in order to avoid memory effects. The position of the needle in the injector has proved crucial for some analytes, probably as a result of the injector not being uniformly heated [214]. Although, as can be seen from Fig. 4.31, this factor has little effect on some analytes, it is always preferable to keep the needle in the same position. As an alternative to thermal desorption, analytes can be desorbed from the fibre by using organic solvents at a special interface and subsequently analysed by HPLC [222].


169

For compounds amenable to HPLC, a combination of this technique and SPE is the preferred choice on account of its operational simplicity and favourable limits of detection. 4.5.4. Analytical features of SPME This section deals with the basic analytical properties enhanced by the use of SPME (viz. selectivity and sensitivity), in addition to other major analytical properties including accuracy and precision.

Sensitivity The amount of analytes that is extracted by SPME depends mainly on the nature of the coating, its volume, whether and how the target analytes are derivatized and the matrix modified, and whether the sample is heated or the coating cooled. The amount of analyte that is absorbed, and hence the limit of detection for the analyte, can be controlled via the phase thickness. Based on eq. (4.45), the amount of analyte extracted by the fibre coating is directly proportional to the coating volume; also, sensitivity improves as the coating volume is increased (by increasing the coating thickness, the fibre length or both). With non-polar and moderately polar organic compounds, conventional coatings are capable of extracting at least a few picograms of analytes from low part-per-trillion samples, a level that constitutes the typical limits of detection specified by regulatory agencies. This amount is sufficient for identification and quantitation with an ion-trap mass spectrometer. Sensitivity in SPME peaks at equilibrium; for practical purposes, however, the extraction time can be shortened to an extent depending on the particular sensitivity requirements.

Selectivity As noted earlier for the extraction of BTX compounds and shown in Fig. 4.28, coating/sample partition coefficients depend largely on the type of coating for a given analyte. Derivatization and temperature also have some influence on selectivity and must be adjusted as a function of the analytes, matrix and state of the sample.

Accuracy and precision Because SPME is primarily an equilibrium technique, it requires calibration when used for quantitation purposes. With air samples, calibration is quite easy [223]. Because sampling occurs either in an open space or in a large volume of air, the amount of analytes extracted by the fibre coating is, based on eq. (4.46), linearly related to the partition coefficient, Kt~, which in turn is determined by moisture and temperature. The concentration of analytes in air can thus be determined from the response of the GC detector after correcting for moisture and temperature. For relatively clean aqueous samples (< 1% organics) such as drinking water or other well-defined matrices, external calibration works very well and is usually carried out by spiking a known amount of the target analytes

170


into a clean matrix and then performing SPME. The analyte concentration in unknown samples can be accurately determined by comparing the detector signal with the calibration curve. Multi-level validation for drinking water revealed a high consistency between the results of SPME and the purge and trap technique [224]. With complex soil samples, external calibration may not work well because of matrix effects, and standard additions or internal standards may have to be used instead [206,210,225]. For internal standards to work well, their partition coefficients must be similar to those of the target analytes. Isotopically labelled analogues of target analytes are the best internal standards for SPME because their chemical and physical properties are similar to those of their unlabelled counterparts [210]. Because spiked analytes may not interact with the matrix as strongly as native ones, the experimental conditions must be carefully adjusted to ensure the effective release of native analytes into the extracting media [222]. Analytes with very large partition coefficients because of their strong affinity for the coating or by effect of the sample being heated simultaneously with cooling of the coating can be thoroughly extracted. Although calibration is no longer necessary, it is usually done anyway in order to confirm the extraction efficiency. Because SPME is a single-step t e c h n i q u e - which minimizes sources of random errors associated with transfer of the analytes it exhibits very good precision with clean, model samples. In fact, the precision of SPME is typically in the region of 5% (as RSD) with manual operation and can be as low as 1% with model samples processed using an auto-sampler. On the other hand, complex matrices and carry-over effects can result in RSD values as high as 92% [226,227].

4.5.5. Advantages and shortcomings of SPME Advantages As a sample preparation technique, SPME is well-suited to the analysis of volatile and semi-volatile compounds in aqueous, gaseous and solid matrices. Because it dispenses with the use of solvents not only in extraction but also during injection it greatly improves the efficiency of chromatographic separations [228]. Solvent-free injection facilitates the use of thinner columns. The cylindrical geometry and small diameter of the fibre can fit into an injector with a very small volume or directly into a column. Analytes can be thermally desorbed very rapidly, thus generating a very narrow band in the column. The use of a column with a small inner diameter and a thin stationary phase improves the separation efficiency and facilitates expeditious GC separations in highly compact instruments. SPME is thus a highly useful sampling tool for on-site environmental analyses when coupled to a portable high-resolution isothermal gas chromatograph. Combined with a smaller injector capable of pulse heating, SPME can be used to insert an extremely narrow band of analytes into a GC column and facilitate separation in a very short time (a few seconds) [229]. All these features make SPME a highly suitable, fast screening technique (especially for field work). SPME is also an excellent tool for extracting compounds amenable to GC, which provides an alternative to well-established sample preparation methods for polar compounds based on on-line SPE-HPLC.


171

Shortcomings"

One of the factors governing SPME performance is the quality of the needles used, which varies among manufacturers. One major problem arises from the fact that the fibres are fragile and readily broken. The GC injector temperature for desorption of the analytes is also a crucial variable and should be maintained below the maximum operating temperature of the coating (viz. 260-320~ depending on the particular phase). Such a temperature should never be exceeded while conditioning the fibre a mandatory operation for each new fibre and for fibres that have not been in use for some time. The time required for thermal conditioning is usually specified by the manufacturer. Even with careful conditioning of the fibre, some bleeding of the stationary phase can occur, however. One other potential problem arises from carry-over of the fibre, which may occur with some analytes (e.g. organochlorine pesticides) and is difficult to suppress even at high desorption temperatures. If the problem exists, the best solution is to perform blank GC runs with a fibre between successive samplings. Samples containing large amounts of suspended matter can result in damage to the fibre coating during agitation. High-molecular weight compounds such as humic acids and proteins can be irreversibly absorbed to the fibre, thus altering the properties of the stationary phase and rendering it useless. This can be avoided by protecting it with a semi-permeable membrane [230]. Irreversible absorption is one possible reason for the poor reproducibility and linearity occasionally encountered in SPME, and so is the formation of gas bubbles on the fibre surface, which is difficult to avoid and affects mass transfer rates. Both problems can be circumvented by using headspace SPME (or the above-mentioned isotopically labelled internal standards) in conjunction with mass spectrometric detection. The former solution is simpler; the latter is certainly more reliable, but also rather expensive.

4.5.6. Applications of SPME The applications of SPME have so far focused on the extraction of organic compounds from various types of matrix including air, water and soil, usually followed by direct transfer into a GC injector for thermal desorption, individual separation on the column and quantitation. In combination with headspace sampling, SPME can also be used to extract organic compounds from wastewater, sludge, soil and other complex matrices [231]. Sub-part-per-billion limits of detection and good precision have been achieved when sampling at room temperature from such complex samples [218]. Even with thin coatings (e.g. 15 ~tm polydimethylsiloxane), SPME affords the extraction of semi-volatile compounds such as PAHs and PCBs [210]. SPME can also be used to extract target analytes from food and drug samples. Thus, it has been employed for the extraction of caffeine from coffee and tea [225], and for that of volatile impurities in drugs. Headspace SPME has also been tested for flavour analysis in foods. Thus, the SPME/GC/TOF-MS tandem was successfully used for the rapid analysis of volatile flavour compounds in apple fruit. The sample (300-450 g of apple) was subjected to static headspace sampling for 4-6 h in order to allow the volatiles

172


to reach the steady state, after which the headspace was sampled with an SPME fibre 1 cm long coated with a 100 pm thickness of polydimethylsiloxane. The subsequent desorption from the fibre, separation and detection allowed the thorough characterization of the unknown compounds. Throughput, variability, linearity, the saturation kinetics and the matrix effects on sampling were examined in depth using aroma standards. Although the total analysis time was claimed to be 10 min - - and hence much shorter than the typical 100-120 min required for purge and trap sampling and subsequent GC-FID m one should also consider the 4-6 h needed for the headspace to reach the steady state [232]. With no steady state allowed, but only 4 rain for creating the headspace distribution, the analysis time was dramatically shortened [233]. A comparison of SPME and the purge and trap technique with both thermal desorption and the direct headspace mode for the removal of volatile organic compounds in environmentally hazardous waste provided similar chromatograms for VOCs processed by SPME and PT (46 compounds were detected in both cases with > 90% spectral mass) but poorer results with direct headspace sampling (only 11 compounds were identified in this case). It should be noted that the direct headspace mode was implemented without the aid of cryogenic cooling, which assists in refocusing the analytes at the column head; by contrast, the PT device was equipped with an electrical cold trap in the desorption chamber that refocused the analytes prior to GC separation. The consistency between the qualitative results of SPME and PT, and the faster performance of the former, led to SPME being recommended for screening of VOCs [234]. Table 4.10 compares the results provided by SPME and the headspace modes for petrochemical wastewater. As can be seen, the headspace-based method was more sensitive (responses were 1.6-7.8 times higher); both, however, were similarly reproducible. Compounds with an increased RSD may require a longer equilibration time. As the molecular size of the analyte increases, the speed of convection of the analyte through the matrix to the fibre decreases and diffu-

TABLE 4.10 COMPARISON OF IMMERSION AND HEADSPACE SPME ANALYSIS OF PETROCHEMICAL WASTEWATER

Compound

Immers ion response (area x 10~')

% RSD (n = 5)

Headspac e response (area x lOt')

% RSD (n = 5)

Benzene Toluene Ethylbenzene m/p-Xylenes o-Xylene 1-Ethyl-2-methylbenzene 1,3,5-Trimethylbenzene 1,2,4-Trimethylbenzene Naphthalene

83.3 343 12.9 259 138 34.9 53.5 15.6 30.2

3.1 5.5 2.0 12 11 13 23 12 22

147 560 101 605 331 115 191 57.5 89.9

6.1 5.3 8.6 3.3 5.1 13 17 15 16


173

sion into the fibre coating becomes slower. The addition of a salt to improve extraction and sensitivity tends to delay equilibration. A compromise between sensitivity and equilibration time may be reached by using a fibre with a thinner coating [234]. The characterization of water-soluble components in slurries is one use of SPME with mixed solid-liquid samples. In one application, dried homogenized solid samples (10 mg of sewage sludge or sediment) were slurried in 4 ml of H20 saturated with NaC1 and adjusted to pH 2 with HCI for extraction for 1-15 h, which was followed by desorption into 4"1 methanol/ethanol over 2 min. The extracted compounds were either injected into a liquid chromatograph or fed directly via an electrospray ionization interface to a mass spectrometer with 1 s m/z scans from 50--700 or selected-ion monitoring. The major components extracted included phthalates, fatty acids, non-ionic surfactants, chlorinated phenols and carbohydrate derivatives [235]. Although SPME is usually employed with organic compounds, it has also been applied to inorganic ones (e.g. in the extraction of inorganic lead and tetraethyllead from water [236]). Also, an interesting method for the determination ofhaloacetic acids in water based on in situ derivatization, headspace separation, SPME and GC/ion-trap MS was recently reported [237] whereby the methyl esters of the analytes, formed by treatment with dimethyl sulphate, were subjected to headspace separation and then to extraction using a Carboxen-polydiethylsiloxane fibre. The method was linear over 2 orders of magnitude, the limits of detection were compound-dependent and ranged from 10 to 450 ng/1, and the results were consistent with those of the EPA recommended method for the analytes.

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Chapter 5

Microwave-assisted solid sample treatment

5.1. INTRODUCTION Whether simply for heating up a meal or preparing a sample for analysis via a multistep process, microwave technology is of considerable significance to both homes and analytical laboratories. In fact, it can help reduce the time taken to develop many processes, so it is of high value with many types of samples. The way in which microwaves enhance some processes is not fully understood, however. The main factors to be considered here include improved transport properties of molecules, molecular agitation, the heating of solvents above their boiling points and, in some cases, product selectivity. Analytical chemists began using microwaves in 1975 to digest biological samples [1]. A domestic microwave oven was then used to rapidly heat a mixture of sample and digestion acids to its atmospheric boiling point in an Erlenmeyer flask. Microwave-assisted methods have been widely used in analytical chemistry ever since their first application was reported. The latest advances in the use of microwaves in various analytical chemical fields include sample digestion for elemental analysis [2], solvent extraction [3], sample drying [4], moisture measurement [5], analyte adsorption and desorption [6], sample clean-up [7], chromogenic reactions [8], solid-phase retention [9], elution [10], distillation [11], microwave plasma atomic spectrometry [12] and synthetic reactions [13], among others. This chapter describes the principal applications of microwaves to the pretreatment of solid samples, with special emphasis on digestion and extraction, which are their two main uses in analytical chemistry. The description is preceded by a discussion of the fundamentals of microwave energy and its interaction with matter, and also of the equipment used by analytical laboratories, which can be of the open or closed type depending on whether they operate at atmospheric pressure or a higher level and whether they use multi-mode or focused microwaves. Selected designs developed for specific purposes are also commented on. Before applications are dealt with, the main variables governing microwave-assisted processes and the parameters characterizing specific microwave treatments are examined. The applications discussed include not only microwave-assisted digestion and extraction which are the two most widely implemented and hence those with the highest potential interest to readers m but also others of special significance to solid sample treatment such as microwave-assisted drying, distillation and protein hydrolysis. Finally, some safety recommendations on the use of microwave equipment are made.

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5.2. FUNDAMENTALS OF MICROWAVE ENERGY AND ITS INTERACTION WITH MATTER Microwave radiation consists of electromagnetic waves and hence of an electric and a magnetic field that are normal to each other. Microwave energy is a non-ionizing type of radiation that causes molecular motion through migration of ions and rotation of dipoles without altering molecular structure. The microwave region of the electromagnetic spectrum lies between infrared light and radio frequencies, and spans the frequency range from 300 MHz to 300 GHz, which corresponds to wavelengths from 1 m to 1 cm. The wavelengths from 1 to 25 cm are extensively used for RADAR (RAdio Detection And Ranging) transmissions and the remaining wave-length range is used for telecommunications. Four specific frequencies, viz. 915 • 25, 2450 • 13, 5800 • 75 and 22 125 + 125 MHz, are used for industrial and scientific microwave heating. These frequencies were reserved for industrial, scientific and medical use by the US Federal Communications Commission (FCC) and conform to the International Radio Regulations adopted in Geneva in 1959. Most domestic microwave ovens operate at 2.45 GHz. The typical energy output of a microwave system is 600-700 W. This allows approximately 43 000 cal to be supplied within 5 min to the microwave cavity for sample heating [14]. There is a clear difference between microwave spectroscopy and microwave dielectric heating effects. Thus, in microwave spectroscopy, molecules are examined in the gas phase and the microwave spectrum for a molecule exhibits many sharp bands [15] over the frequency range 3-60 GHz. Such sharp bands arise from transitions between quantized rotational states of the molecules. Microwave spectroscopy provides an excellent fingerprinting method for identifying molecules in a gas phase and has been used, for example, to confirm the presence of a wide range of molecules in outer space. The lifetime of the excited state produced by a particular rotational state in a gas phase at a low pressure is long. However, by virtue of Heisenberg's uncertainty principle, at pressures in the region of 10 -~ mm Hg, the lifetime is reduced by collisions and spectral peaks broaden as a result. In liquids and solids, where molecules are generally not free to rotate separately, spectra are too broad to be observed. It is to these phases that microwave dielectric loss heating effects are relevant as distinguished from spectroscopic effects of microwave energy. A material can be heated by applying energy to it in the form of high-frequency electromagnetic waves. The origin of the heating effect is the ability of an electric field to exert a force on charged particles. If the particles present in a substance can move freely through it, then a current has been induced. On the other hand, if the charge carriers in the substance are bound to certain regions, they will move until a counterforce balances them, the net result being dielectric polarization. Both conduction and dielectric polarization are sources of microwave heating. The heating effect of microwaves depends on their frequency and on the applied power; unlike microwave spectroscopy, however, the effect does not result from well-spaced discrete quantized energy states.


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5.2.1. Dissipation factor The dielectric properties of materials are defined by two different parameters, namely: the dielectric constant and the dielectric loss. The dielectric constant, e, describes the ability of a molecule to be polarized by the electric field. At low frequencies, e reaches a maximum as the maximum amount of energy can be stored in the material. The dielectric loss, e', measures the efficiency with which the energy of the electromagnetic radiation can be converted into heat. The dielectric loss goes through a maximum as the dielectric constant falls [16]. The dissipation factor (tan 6) is the ratio of the dielectric loss of the sample, also called "loss factor", to its dielectric constant" tan 6 = e'/e. When microwave energy penetrates a sample, the energy is absorbed by the sample at a rate dependent on its dissipation factor. Penetration is considered to be infinite in materials that are transparent to microwave energy and zero in reflective materials (e.g. metals). The dissipation factor is a finite amount for absorptive samples. Because the energy is rapidly absorbed and dissipated as microwaves pass into the sample, the greater the dissipation factor of the sample is, the less microwave energy at a given frequency will penetrate into it. One effective way to characterize penetration is via the half-power depth or the distance from the sample surface at which the power density is reduced to one-half that at the surface [17]. The half-power depth varies with the dielectric properties of the sample and also, roughly, with the inverse of the square root of the frequency. 5.2.2. Transformation of microwave energy into heat Microwave energy is lost to the sample via two mechanisms: ionic conduction and dipole rotation. Both occur simultaneously in many practical applications of microwave heating. Ionic conduction is the conductive migration of dissolved ions in the applied electromagnetic field. This ion migration is a flow of current that results in /eR losses (heat production) due to resistance to ion flow. All ions in a solution contribute to the conduction processes; however, the fraction of current carried by any given species is determined by its relative concentration and its inherent mobility in the medium. Therefore, the losses due to ionic migration depend on the size, charge and conductivity of the dissolved ions, and are subject to the effects of ion interaction with the solvent molecules [18]. Ion conduction is influenced by the ion concentration, ion mobility and solution temperature. Every ionic solution contains at least two ionic species and each species will conduct current according to its concentration and mobility. The dissipation factor of an ionic solution changes with temperature, which affects ion mobility and concentration. Dipole rotation refers to the alignment, by effect of the electric field, of molecules in the sample that have permanent or induced dipole moments. As the electric field of microwave energy increases, it aligns polarized molecules. As the field decreases, thermally induced disorder is restored. In fact, applied microwave fields cause molecules, on average, to temporarily spend very slightly more time pointing in one direction than in others. Associated with that very small fraction of preferred orientation there is another very small fraction of molecular order imposed and hence a tiny bit of energy. When the

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field is removed, thermal agitation returns the molecules to disorder, during relaxation time, and thermal energy is released. At 2450 MHz, the alignment of the molecules followed by their return to disorder occurs 4.9 • 10`7 times each second and results in very rapid heating. However, the efficacy of heating by dipole rotation depends upon the characteristic dielectric relaxation time of the sample, which in turn is a function of its temperature and viscosity. The dielectric relaxation time is the time taken by the molecules in the sample to achieve 63% of their return to disorder. The maximum energy conversion per cycle for many materials (dielectric loss due to dipole rotation) will occur when to = l/-r, where to is the angular frequency of the microwave energy (in rad/s) and -r the dielectric relaxation time of the sample [19]. A non-ionic polar sample with a 1/'r value close to the angular frequency of the input microwave energy will possess a high dissipation factor. By contrast, when 1/'r for the sample is substantially different from the microwave frequency, the sample will have a low dissipation factor. Temperature dictates to a great extent the relative contribution of each of the two energy conversion mechanisms (dipole rotation and ionic conduction). With small molecules such as water and other solvents, the dielectric loss to a sample due to the contribution of dipole rotation decreases as the sample temperature increases. By contrast, the dielectric loss due to ionic conduction increases as the sample temperature increases. Therefore, as an ionic sample is heated by microwave energy, the dielectric loss to the sample is initially dominated by the contribution of dipole rotation. As the temperature increases, the dielectric loss is dominated by ionic conduction. The relative contribution of these two heating mechanisms depends on the mobility and concentration of the sample ions and on the relaxation time of the sample. If the ion mobility and concentration of the sample ions are low, then sample heating will be entirely dominated by dipole rotation. On the other hand, as the mobility and concentration of the sample ions increase, microwave heating will be dominated by ionic conduction and the heating time will be independent of the relaxation time of the solution. As the ionic concentration increases, the dissipation factor will increase and the heating time decrease. The heating time depends not only on the dielectric absorptivity of the sample but also on the microwave system design and the sample size.

5.2.3. Microwave heating There is a clear difference between conductive and microwave heating: because the vessels used in conductive heating are usually poor conductors of heat, they take time to heat and transfer such heat to the solution. Also, because the liquid vaporizes at the surface, a thermal gradient is established by convection currents so only a small portion of the fluid is at the temperature of the heat applied to the outside of the vessel. Therefore, with conductive heating, only a small portion of the fluid is above the boiling point of the solution. On the other hand, microwaves heat all of the sample fluid simultaneously without heating the vessel. Therefore, with microwave heating, the solution reaches its boiling point very rapidly. Because the rate of heating is so much more rapid, substantial localized superheating can occur [14]. Table 5.1 shows the effect of microwave heating on several solvents [20].


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TABLE 5.1 TEMPERATURE REACHED BY VARIOUS SOLVENTS UPON HEATING FROM ROOM TEMPERATURE USING MICROWAVE ENERGY OF 2450 MHz AND 560 W FOR 1 MIN Solvent

T, ~

Boiling point,~

Water Methanol Ethanol 1-Propanol 1-Butanol 1-Pentanol 1-Hexanol l-Chlorobutane 1-Bromobutane Acetic acid Ethyl acetate Chloroform Acetone Dimethylformamide Dimethyl ether Hexane Heptane Carbon tetrachloride

81 65 78 97 109 106 92 76 95 110 73 49 56 131 32 25 26 26

100 65 78 97 117 137 158 78 101 119 77 61 56 153 35 68 98 77

5.3. MICROWAVE EQUIPMENT Microwave equipment used for sample treatment can be classified into two groups according to the way microwave energy is applied to the sample, namely: multi-mode systems, where the microwave radiation is allowed to randomly disperse in a cavity, so every zone in the cavity and the sample it contains is evenly irradiated; and single-mode or focused systems, where the microwave radiation is focused on a restricted zone where the sample is placed for subjection to a much stronger electrical field than in the previous case. Usually, multi-mode systems are of the closed-vessel type, and focused systems of the open-vessel type [21], the former being defined as systems where the microwave treatment is conducted at a high pressure and the latter as those where microwaves are applied at atmospheric pressure, so the system is subject to no overpressure. This mutual association, however, is incorrect as some commercial devices that operate at a high pressure use focused microwaves [22,23] and domestic ovens have been used to couple microwave treatment to detection by using multi-mode sample irradiation but at atmospheric pressure [24,25]. The latter application has aroused much confusion as many authors consider these systems, which are usually flow-injection manifolds, to be of the closed-vessel type [26]. In a different context, this definition is quite correct and causes no confusion as the sample, once inserted into the flow-injection system, actually

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Fig. 5.1. Schematic of a magnetron. circulates through a series of tubes and devices isolated from the outside. However, in order to avoid confusion, we shall use the designation "closed-vessel" in this chapter only to refer to systems operating above atmospheric pressure. 5.3.1. Main components of a microwave device

Both multi-mode and focused microwave devices consist of four major components, namely: (a) The microwave generator, usually called the "magnetron", where the microwave energy is produced; (b) The waveguide, which is used to propagate the microwaves from the magnetron to the microwave cavity; (c) The applicator, where the sample is placed; (d) The circulator, which allows microwaves to pass only in the forward direction. Basically, the magnetron consists of a number of identical resonators (small cavities) arranged in a cylindrical pattern around an also cylindrical cathode (see Fig. 5.1). A permanent magnet is used to produce a strong magnetic field normal to the cross-section. The anode, which consists of a series of circuits tuned to oscillate at a specific frequency (or its overtones) is kept at a high positive voltage relative to the cathode. Electrons emitted from the cathode are accelerated towards the anode block, but the presence of the magnetic field produces a force in the azimuthal direction that causes the electron trajectory to be deflected in the same direction. As the deflected electrons pass through


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the resonator gaps, they induce a small charge into the circuit that causes the resonator to oscillate. Alternate cavities are linked by two small wire straps (mode straps) that ensure the correct phase relationship. This process continues until the oscillation has reached a sufficiently high amplitude. At that point, the radiation is taken off the anode via an antenna enclosed in the vacuum envelope of a tube. In microwave systems used for sample preparation, the power output of the magnetron is controlled by "cycling" the magnetron to obtain an average power level. The duty cycle is the time the magnetron is "on" divided by the time base. For example, a time "on" of 5 s with a time base of 10 s is a duty cycle of 0.5, and so is a time "on" of 0.5 s with a time base of 1 s. The time base for the magnetron in microwave systems used for sample preparation is 1 s. Thus, in order to obtain one-half the rated output of 600 W, the magnetron would be "on" for 0.5 s and "off" for 0.5 s. Appliance-grade microwave ovens typically have a time base of 10 s. This relatively long time is not desirable for heating analytical samples because heat losses can be significant during the long "off" time (5 s for a duty cycle of 0.5). The power output of a magnetron can be affected by overheating resulting from reflected microwaves. Reflected power occurs when the travelling electromagnetic waves are reflected and the flow of energy is partly in the reverse direction: towards the magnetron. Methods that transfer travelling waves from one medium to another without reflectance are called "impedance matching methods". If the microwaves travel from the magnetron to the sample and are not reflected, then the system is perfectly matched. If there is reflection, then the system is said to be mismatched. Mismatching may cause overheating of the magnetron, a loss in output power or even destruction of the magnetron. The waveguide is a rectangular or circular channel constructed of reflective material such as sheet metal and is designed to direct the microwaves generated by the magnetron to the cavity without a mismatch. Waveguides with a rectangular cross-section are the more widely used. They are available in sizes for use at frequencies from 320 MHz to 333 GHz. The minimum frequency that can be propagated through the waveguide is related to the dimensions of the rectangular cross-section by the expression c/f = 2d, where c is the speed of light, f the cut-off frequency and d the larger of the dimensions of the rectangular cross-section. The applicator is the zone in which microwaves are applied to the sample. It can be a multi-mode cavity where microwaves are randomly dispersed or the waveguide itself, in which case the sample vessel is placed directly inside it to focus the microwave radiation onto the sample. The walls of a microwave cavity should be reflectant in order to prevent leakage of radiation and increase the efficiency of the oven. The pathway followed by the microwaves is well defined into recognizable patterns or modes that have a beginning and an end. The direction and number of excitation modes in a cavity of a certain size are two factors to be considered with a view to ensuring uniform microwave heating. There is rarely a perfect match between the frequency used and the resonant frequency of the load, so if the energy is reflected by the walls, the absorbance is increased because the energy passes through the sample more often and can be partially absorbed on each passage. A sample in the microwave cavity that has a high dissipation factor could be a model of a perfectly matched microwave system by absorbing nearly 100% of the input power. If too much

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Fig. 5.2. Major components of a typical multi-mode microwave system.

energy is reflected back into the waveguide, a mismatch between the magnetron and the microwave occurs that can alter the output and result in damage to the magnetron through overheating. Uniform distribution of microwave radiation in a microwave cavity can be achieved by using a mode stirrer, which is a fan-shaped blade that is used to reflect and mix the energy entering the microwave cavity from the waveguide. A mode stirrer assists in distributing the incoming energy so that heating of the sample will be more independent of position. Figure 5.2 depicts a typical multi-mode microwave system. Unlike microwave cavity systems, in focused microwave systems a single vessel is placed directly in a microwave waveguide that acts as the applicator (see Fig. 5.3). The bottom few inches of the vessel are directly exposed to the microwaves, whereas the upper region of the vessel remains cool. This results in an effective condensing mechanism inherent in the design. While vessels are open to the atmosphere, the refluxing action minimizes losses of solvent and some volatiles. Vessel openings have been designed to allow automated reagent addition and restrict contamination from the atmosphere. Most commercial ovens, whether single-mode or multi-mode models, are protected from potential damage caused by reflected energy by an automatic cut-off mechanism; also, some ovens provide additional protection in the form of a circulator that directs reflected energy into a dummy load. A circulator is essentially a device that uses ferrites and static magnetic fields to allow microwaves to pass in the forward direction but diverts reflected waves into a dummy load where the energy is harmlessly dissipated as heat.

5.3.2. Closed-vessel microwave systems The earliest microwave systems for analytical purposes were closed-vessel systems with a multi-mode cavity. The primary improvement over domestic units was the addition


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REFRIGERANT

o

SOLVENT RECONDENSATION

MAGNETRON

Y

l CELLULOSE CARTRIDGE

~ FOCUSED MICROWAVES

SAMPLE

O1 MAGNETIC STIRRER

Fig. 5.3. Scheme of a focused microwave system. (Reproduced with permission of Springer-Verlag.) of safety mechanisms. The early units, though built from domestic-oven cavities and doors, isolated and ventilated the cavity to prevent acid fumes from attacking the electronics. Ever since the first commercial laboratory microwave unit (Model MDS-81 D from CEM Corporation) was introduced in 1985 [27], a number of manufacturers have delivered gradually improved versions with more uniform microwave fields, the ability to control the microwave power and, most importantly, increased safety. In the mid-1980s, some researchers started to build or adapt temperature- and pressuremonitoring equipment for use inside a microwave cavity. The primary challenges were to develop probes that would not disturb the microwave field and to construct wavelength attenuator cut-offs for the probes so that they could enter the microwave region while preventing microwaves from leaving the microwave cavity. Monitoring temperature and/or pressure during digestions or extractions started the age of controlled microwave treatments, the study of the mechanisms of microwave-assisted processes and the development of transferable standard microwave sample preparation methods. These developments

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promoted an outgrowth of microwave use that was reflected in a substantial increase in microwave sample preparation publications. The earliest commercial laboratory microwave units with pressure and temperature feedback control, which were developed in 1989 and 1992, respectively, afforded a more rigorous design and control of microwave sample preparation procedures. Concurrent with these developments, the microwave vessel also evolved significantly. The vessels in microwave systems are relatively transparent to microwave radiation and inert to the reagents used. The materials in contact with the sample are usually of fluoropolymer, quartz or glass composition. Closed, pressurized vessels are frequently multi-layered, with a structural outer casing of a microwave-transparent polymer such as polyetherimide. This polymer is commonly used for its tensile strength, rigidity and ability to contain accumulated pressure. Closed microwave vessels exist in two different forms. One encompasses uninsulated, relatively thin, single-walled fluoropolymer vessels and fluoropolymer vessels of the same material with a thin outer layer made of polyetherimide or rigid composite plastic. These vessels have minimal insulating characteristics and allow large amounts of heat to escape. The other type of closed vessel is a well-insulated container, usually of very thick-walled fluoropolymer, or one with a very thick outer layer or casing (or both). These vessels retain heat in a highly efficient manner, so they do not allow rapid cooling by ambient air forced over them within the microwave cavity [16]. Commercially available closed vessels can be chronologically divided into three generations. The first generation was an all-Teflon [polytetrafluoroethylene (PTFE)] design with low-pressure limits that decreased as the vessel aged through stress from previous treatments The second generation was that of jacketed vessels, which typically consisted of a Teflon liner and cap on a polymer case (usually of polyetherimide) that increased the pressure limit of the vessel up to 20 atm. The third generation of vessels, also lined, are thoroughly redesigned models that can withstand pressures as high as 60-110 atm. Figure 5.4 depicts one of the most recent (third-generation) designs of high-pressure closed vessels, together with a typical high-pressure module where up to ten vessels can be placed for simultaneous sample pretreatment. This type of high-pressure vessel was designed by OI Analytical Corporation for maximum safety by incorporating a dual pressure-relief mechanism. This design includes a safety rupture membrane for overpressure venting at more than 600 psi and a break-away safety disc for rapid pressure relief at more than 1000 psi. The type of design and, especially, the material used to construct closed vessels, should be chosen as a function of the type of treatment to be applied and the subsequent type of analysis to be performed on the sample. For example, materials such as PFA (a perfluoroalkoxy resin) and TFM [a copolymer of tetrafluoroethylene and a small amount of perfluoro(propyl vinyl ether)] should be used in the microwave digestion of samples prior to ultratrace analysis, where the material should be inert to hot concentrated acids and contain minimal amounts of leachable contaminants [28]. Raster electron microscopy has shown PFA to be the most suitable material for this purpose; in fact, its surface was found to remain virtually unaltered even after 100 digestion cycles [29].


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Fig. 5.4. (A) Elements of a high-pressure vessel. (B) Arrangement of the vessels in a high-pressure module.

190



191

Focused closed-vessel microwave devices

Most commercially available closed-vessel microwave systems are based on multimode microwaves; however, the advantages of high-pressure vessels and focused microwave heating have led to the development of systems that combine both assets. These so-called "focused high-pressure, high-temperature microwave systems" [30,31 ] consist of an integrated closed vessel and a focused microwave-heated system operating at a very high pressure and temperature. One particular system uses a rectangular brass waveguide to direct and focus the microwaves by means of the antenna coupler and a polymer bomb to contain the sample and solvents, and also a special configuration of antennas is used: internal and external antenna systems are carefully sized to achieve efficient coupling of microwave energy between the magnetron and the sample and solvents. This design provides highly efficient (> 90%) energy transfer from the microwave generator to the sample and solvents, and can be used at both low and high powers. Recently, an extension of the above-described focused single-closed vessel microwaveheated system was developed that allows the simultaneous treatment of up to six samples in a gas-pressurized metal chamber while controlling and measuring the pressure in one vessel. Pressures up to 130 bar and temperatures up to 320~ can thus be reached [32]. Dynamic closed-vessel microwave systems

Dynamic systems for high-pressure microwave treatment were developed much later than open-vessel systems. Operating under a high pressure reduces the flexibility afforded by working at atmospheric pressure. However, some recently developed devices allow microwave-assisted high-pressure digestion and extraction in a dynamic manner [33,34]. In 1999, a microwave-assisted flow digestion system was developed from a novel pressure equilibration system that affords temperatures up to 250~ and pressures in the region of 35 bar with previously slurried solid samples. Figure 5.5A depicts the flow microwave digestion system and illustrates the principle of pressure equilibration. The equipment consists of a pressurized reactor vessel that can be made of PTFE or PFA. The heating zone, located in the microwave field of a waveguide, is made of alumina ceramics and the cooling zone is made of poly(ether-ether ketone) (PEEK"). Both parts are connected in a gas-tight manner. The reaction coil, which consists of PTFE or PFA tubing, is placed within the heating zone of the reactor. The length of the digestion tube is restricted by the dimensions of the waveguide: the longer the digestion tube is, the higher is the reactive volume within the microwave field and the higher the sample Fig. 5.5. (A) Scheme of a flow digestion system and the principle of pressure equilibration: A pressure reactor, B heating zone, C cooling zone, D digestion coil, E cooling device, F connection for gas supply, G restrictor tube, H collector vial, I temperature sensor, J high-pressure pump, K injection valve, L sample loop, M sample, N and O peristaltic pumps. (Reproduced with permission of the American Chemical Society.) (B) Manifold for dynamic microwave-assisted extraction: 1 solvent, 2 pump, 3 microwave oven, 4 extraction chamber, 5 temperature set-point controller, 6 thermocouple, 7 fluorescence detector, 8 recording device, 9 restrictor, 10 extractor. (Reproduced with permission of Elsevier.)

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throughput as a result. Directly outside the microwave field, the temperature of the reaction mixture is measured by means of a Pt sensor. The cooling device consists of a water-cooled titanium rod wrapped with Teflon tubing. The reaction tube ends open at the funnel-shaped bottom of the reactor vessel, which is connected to a nitrogen bomb and supplied with N 2 at a constant pressure up to 5 bar. A restrictor tube is connected gas-tight to the bottom of the reactor vessel, so there is a small continuous gas flow through it. The high pressure within the reactor causes the boiling temperature of the reaction mixture following passage through the digestion coil to rise without exerting mechanical strain on the tube walls. This is the result of the pressure balance inside and outside the reaction tube. It is necessary to supply enough microwave energy to reach the boiling point inside the reaction tube. At the boiling stage, vapour bubbles are formed that can no longer absorb microwave energy; the vapour condenses and microwave energy is absorbed again, so a boiling equilibrium is maintained; in that way, the temperature is controlled only by the applied pressure. The most salient advantage of this pressure equilibrium system is that, even with extremely fast oxidation reactions (e.g. the digestion of glucose), the pressure inside and outside the reaction tube remains identical and the tube does not burst [33]. Recently, Ericsson and Colmsj6 [34] developed a dynamic high-pressure microwaveassisted extraction device that allows the treatment of solid samples while avoiding the principal drawback of the previous approach (viz. the relative complexity of the assembly and the inability to directly introduce unslurried solid samples). The experimental set-up used is shown in Fig. 5.5B. The extraction cell is made of PTFE and has an inner volume of 9 ml. It is designed to fit a Soxhlet-type extraction thimble made of cellulose. The tubes and the fingertight fittings are made of PEEK". During extraction, fresh solvent is continuously pumped through the extraction cell, which is maintained at a slight overpressure by using a fused silica restrictor in order to keep the solvent in the liquid state. This system allows the extraction to be coupled to a subsequent stage of the analytical process by connecting the extractor outlet to the unit concerned. Figure 5.5B illustrates one possible coupling: connecting the extractor to a fluorimetric detector allows the kinetics of the extraction of PAHs from sediments to be monitored [34].

5.3.3. Open-vessel microwave systems The first completely re-engineered laboratory focused microwave system was introduced by Prolabo in 1986. Most commercial open-vessel microwave systems manufactured since then are of the focused-microwave type, i.e. they use the waveguide as a single-mode cavity [35]. In these systems, the waveguide is closed at both sides and an opening (typically 42 mm wide) is used to facilitate the incorporation of a flask into it. The opening is located in such a way that the axis of the flasks corresponds to an antinode of the waves transmitted by the waveguide. Precise positioning of the wave antinode is achieved by carefully placing the operating and length selection of the waveguide along with an adjustment of a tuning device (e.g. two metal rods). The electromagnetic energy and the sample are thus highly efficiently coupled and a high power density obtained as a result. According to Prolabo, the coupling efficiency is increased by a factor of 10 relative to the use of a multi-mode cavity. Propagation of microwaves


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outside the waveguide is easily prevented by using a tubular cylindrical stack around the portion of the flask. This arrangement allows the user to add reagents or fresh solvent during the process or to connect a device such as a reflux system. Even though the principle of focused microwaves is efficient in terms of energy transfer, it has allowed for the use of only one flask at a time in most designs. Automation in some systems can enable the sequential use of flasks, but this characteristic can be viewed as a constraint. One recent development involves the use of four flasks at a time by symmetrically splitting the microwave energy among the flasks at the end of each waveguide. In these open devices, a continuously adjustable percentage of the maximum power is used over a given period of time. This result differs from a multi-mode cavity, where pulsed power is predominantly used. The maximum power is in the 200-800 W range, depending on the type of microwave system used [16]. A study about the energy transfer in a focused microwave system revealed that it is possible to accurately predict the temperature of different liquids, whether alone or in the presence of small amounts of samples in an open microwave device. The ensuing model shows that the sample receives much more energy than the useful energy, E u, which is the energy used by the sample to increase its temperature. It also provides an indication of the relative significance of conduction, convection and radiation heat transfer mechanisms. While conduction flow can be considered to be a source of loss for microwave heating, the inverse mechanism is observed in conventional heating techniques. Convective and radiative losses are more important for glass than for the sample because the glass temperature always remains below the sample temperature, whereas glass has no "limiting" temperature such as a boiling point. These results can be used to improve the waveguide and the glass vessel configuration in order to minimize heat losses. The model also provides a numerical estimation of the microwave energy lost in relation to the applied power. This energy loss includes both waveguide loss and glass absorption. The different reagents studied seemingly absorb different amounts of energy under identical operating conditions (given power and time) because of their differential heat capacities and densities. However, different relative heat flows remain similar because they are related to the geometry of the thermal system, which is constant. It has been possible to build abacus for predicting the useful time for a given temperature. Highly precise operating procedures can thus be developed by selecting appropriate time and power settings. Similar abacus could be established for other reagents or organic solvents in order to develop new sample preparation procedures or control product degradation in organic synthesis reactions. Such abacus can also be used to calibrate or check the magnetron [36]. In general, all non-metal vessels used in open-vessel treatments involving conductive heating (e.g. glass beakers, boiling flasks, refluxing containers) can be used in openvessel treatments with microwave heating. The development of new vessels for specific applications in microwave pretreatments is only limited by the analytical chemist's ingenuity. In most open-vessel treatments, it is desirable to achieve as much refluxing as possible so that the need for continual additions of solvent to maintain the volume is decreased. Refluxing is generally better in open-vessel microwave treatments because the vessel is

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secondarily heated as heat from the solution is dissipated and remains cooler. Therefore, with good air flow through the cavity, as is the case with analytical microwave systems, refluxing is more efficient. As noted earlier, not all open-vessel systems (viz. those that operate at atmospheric pressure) are of the focused type. A number of reported applications use a domestic multi-mode oven to process samples for analytical purposes, usually with a view to coupling the microwave treatment to some other step of the analytical process (generally the determination step). Below are described the most common on-line systems used so far, including domestic ovens (multi-mode systems) and open-vessel focused systems, which operate at atmospheric pressure and are thus much more flexible for coupling to subsequent steps of the analytical process. On the other hand, the increased flexibility of open-vessel systems has promoted the design of new microwave-assisted sample treatment units based on focused or multi-mode (domestic) ovens adapted to the particular purpose. Examples of these new units include the microwave-ultrasound combined extractor, the focused microwave-assisted Soxhlet extractor, the microwave-assisted drying system and the microwave-assisted distillation extractor, which are also dealt with in this section. Finally, the usefulness of the microwave-assisted sample treatment modules incorporated in robot stations is also commented on, albeit briefly as such devices are discussed in greater detail in Chapter 10. Open-vessel microwave-assisted on-line sample treatment

Most on-line procedures involving microwaves that are conducted with a view to coupling a microwave treatment (usually digestion) with a detector (usually of the atomic type) for the determination of analytes use either a domestic oven [37-40] or a commercial focused system [41-43] plus appropriate connections. Usually, the coupling is done by inserting a Teflon coil in the oven in order to circulate the suspended solid sample to be subjected to the microwaves [44]. Some systems use domestic or commercial focused systems where the solid sample is directly placed in the sample vessel [45] and an aspiration system is used after the microwave treatment to transfer the extract to the determinative instrument used [37,46] or to an apparatus employed in some other step of the analytical process [40,43]. Ever since Burguera et al. reported the first instance of coupling of a microwave oven to a flow injection (FI) system for solid sample treatment in 1988 [37], a host of applications have been developed to exploit the many advantages of these on-line systems over off-line methods; such advantages include automatability, reductions on expensive time delays between sample delivery and analysis, protection of samples from ambient agents and improved personal safety. Basically, most such applications use an FI system where the sample is introduced as a stabilized slurry; the system includes a reaction coil that is inserted in a microwave oven to perform a sample treatment which, as noted earlier, is usually a digestion [24,41,44,47-52] but can also be a leaching operation [40,53]. The FI system allows the solution or the resulting extract to be transferred to an appropriate detector in order to determine the target species after the microwave treatment. The sample digestion throughput can be improved by using several coils simultaneously in the oven [48]. Microwave ovens have so far been coupled to detectors based on spectrophotometry

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Fig. 5.6. (A) Flow-injection manifold for the wet digestion of shellfish slurry samples: C carrier solution, P peristaltic pump, IC injector commutator, W waste, r restrictor. (B) Dynamic manifold for the analysis of sludge slurries in an on-line digestion-reduction system by FI-HG-AFS: IV injection valve, AFS atomic fluorescence spectrometer, P peristaltic pump. (Reproduced with permission of the Royal Society of Chemistry.)

196


[38,48,50], flame atomic absorption spectroscopy (FAAS) [46,54], electrothermal atomic absorption spectroscopy (ETA-AAS) [39], inductively coupled plasma-atomic emission spectroscopy (ICP-AES) [41,51] and inductively coupled plasma-mass spectrometry [49]; and with derivatization reactors in techniques such as hydride generation-atomic absorption spectrometry (HG-AAS) [47], hydride generation-atomic fluorescence spectroscopy (HG-AFS) [44] and cold vapour-atomic fluorescence spectroscopy (CV-AFS) [52]. In each coupled system, an FI manifold was used to merge the sample with the required reagents (usually acids) prior to the microwave treatment or to add the reagents, separation devices or components needed to derivatize the sample prior to analysis. Figure 5.6 illustrates two examples of coupled systems. That in Fig. 5.6A is a straightforward system where the FI manifold serves the sole purpose of allowing the sample and acid to merge prior to reaching the microwave oven and where the solution resulting from the digestion process is transferred to the autosampler of an ETA-AAS assembly [39]. The system of Fig. 5.6B, which is much more complex than the previous one, includes a phase separator, a hygroscopic membrane and a fibreglass filter to enable the determination of species by AFS following digestion of the sample [44]. The main problem with these systems is the inherent inhomogeneity of the microwave power distribution within the oven cavity: with only a small area occupied by the reaction coil, a high proportion of the microwave power within the cavity is not absorbed. Thus, the positioning and distribution of the reaction coil within the cavity has been identified as an important parameter in achieving uniform heating of the sample and reagents with a view to ensuring efficiently controlled digestion. In addition to being inefficient, the high proportion of unabsorbed power probably reduces the lifetime of the oven. Any resulting heating of the magnetron may in fact reduce the microwave output. Some methods use a domestic or commercial focused microwave oven into which the sample is not introduced as a slurry but rather placed in an appropriate vessel that is then introduced into the oven [37,40,43,45,46]. Figure 5.7 shows two such systems using a domestic microwave oven where, as in the previous case, the microwave treatment was coupled to a subsequent step via an FI system. The system of Fig. 5.7A allows the simultaneous treatment of several samples. It consists of a series of test tubes closed in a glass container that is coupled to a water pump connected to an FI manifold via a selective aspiration system. As can be seen in the figure, while the system affords the simultaneous processing of several samples, it requires up to 14 valves to couple digestion with AAS determination, which results in an extremely complicated set-up [37,46]. On the other hand, the system of Fig. 5.7B is much simpler but can only process one sample at a time. The filter at the extractor outlet prevents clogging of some elements of the FI system by the colloid fraction of the solid sample. As can be seen, these systems possess the same flexibility as the previous ones in addition to that inherent in the FI system which, as noted earlier, is only limited by the user's ingenuity. This system includes some elements such as a membrane phase separator and a mini-sorption column that enable partial automation of the process [40]. In some commercially available focused microwave systems, the sample is placed directly in the vessel [45] or in an extraction cartridge that is in turn placed in the vessel [43]. Figure 5.8 depicts two such systems. In the example of Fig. 5.8A, the microwave system was not coupled to a subsequent step of the analytical process; rather, a dynamic


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Fig. 5.7. (A) Assembly used in the determination of cadmium and zinc in kidney and liver tissue by simultaneous microwave-assisted acid digestion of 6 samples, FI and AAS: CT collector tube, FS flowing sample collector, R recorder. (B) Experimental design for the continuous microwaveassisted leaching, liquid-liquid extraction, sorption/clean-up of phenol compounds in soil samples: IV, injection valve, PS membrane phase separator, o.p. and a.p. organic and aqueous phase, W waste. (Reproduced with permission of Elsevier.)

198



199

system was designed with a view to allowing several consecutive extraction cycles to be performed in order to ensure quantitative extraction of the analytes [45]. Addition of the extractant (a nitric acid solution) and aspiration of the extract after each cycle were done by means of a two-channel piston pump. The implementation of successive cycles afforded quantitative extraction of the analytes, which proved impossible in a single cycle. The sample was placed directly in the extraction vessel and the extract provided by each cycle was aspirated through a cellulose filter to retain solid particles and obtain a clean extract. The use of the pump itself as the interface between the extractor and an FI manifold (Fig. 5.8B) allowed microwave-assisted extraction to be coupled to filtration, preconcentration, individual chromatographic separation and UV detection steps with a view to automating the whole analytical process [43]. Extraction was done by supplying an appropriate extractant (water) in an automatic manner by means of the piston pump. Then, the sample was irradiated with microwaves for a preset time and the extract aspirated through the other pump channel to a vessel connected to the FI manifold, where the analytes where filtered, preconcentrated and chromatographically determined in an automatic manner. Special open-vessel microwave systems Microwave-ultrasound combined extractor The microwave-ultrasound combined system was constructed from a 2.45 GHz, 0-300 W Prolabo Maxidigest 350 single-mode microwave oven; the sample solution was held in a double-walled open 20-150 ml borosilicate reactor with a reflux column at the top. A cup-horn Branson Sonifier 250 at the base of the microwave oven was used for indirect ultrasonic agitation of the sample at 20 kHz via 200 ml of decalin held between the reactor walls. The ultrasonic probe was placed distant enough from the electromagnetic field to avoid interactions and shortcircuits. The microwave system was used to dissolve 0.5 g of Co304 by charring with 20 ml of 16 M HNO 3 and oxidation with 2 ml of H202 for 3-10 min at a microwave power of 150 and 290 W, respectively. The digestion time in the presence and absence of ultrasonic agitation was 1 and 3 h, respectively. The system was also used to digest 1 g of olive oil for the subsequent determination of Cu by charring with 20 ml of HNO 3 for 27 min and oxidation with 3 ml of H202 for 3 min at a microwave power of 180 and 290 W, respectively. The digestion time with and without ultrasonic agitation was 30 and 45 min, respectively [55]. Focused microwave-assisted Soxhlet extractor The first prototype of a focused microwave-assisted Soxhlet extractor was designed by Luque de Castro et al. and

Fig. 5.8. (A) General scheme of a dynamic focused microwave-assisted extractor. (B) Experimental set-up used to integrate microwave-assisted extraction with the subsequent steps of the analytical process. (1) Leaching step: CT controller, MO microwave oven, S sample, R condenser, WR water reservoir, TCPP two-channel piston pump, ER extract reservoir, SV switching valve. (2) Cleanup/preconcentration step: M methanol, A air, B buffer, PP peristaltic pump, F filter, EL elution loop, MC mini-column, R retention direction, E elution direction, IVI-IV3 injection valves, W waste. (3) Individual separation-detection step: HPIV high-pressure injection valve, AC analytical column, DAD diode array detector, SR solvent reservoirs.

200


Fig. 5.9. (A) Conventional Soxhlet extractor. (B) Focused microwave-assisted Soxhlet extractor from Prolabo. (Reproduced with permission of Elsevier.)

constructed by Prolabo (Paris, France) in 1997 [56]. This prototype combines the advantages of the Soxhlet extractor with those of the microwave-assisted process. The extractor design is based on the same principles as a conventional Soxhlet extractor (Fig. 5.9A) modified to facilitate accommodation of the sample cartridge compartment in the irradiation zone of a microwave oven. The latter was also modified by making an orifice at the bottom of the irradiation zone, thus enabling connection of the cartridge compartment to the distillation flask through a siphon as illustrated in Fig. 5.9B. The use of this device has provided better results than conventional Soxhlet extraction in much shorter periods of time [57-60]. The device, which enables focused microwave-assisted Soxhlet extraction (FMASE), retains the advantages of conventional Soxhlet extraction while overcoming restrictions such as the long extraction time and non-quantitative extraction of strongly retained analytes due to the easier cleavage of analyte-matrix bonds by interactions with focused microwave energy, unavailability for automation and the large volumes of organic solvents wasted unlike a conventional Soxhlet extractor, the microwave-assisted system allows recycling of up to 75-85% of the total extractant volume. In addition, solvent distillation in the FMAS extractor is achieved by electrical heating, which is independent of the extractant polarity, thus avoiding the principal problem of commercial focused microwave devices such as those of the Soxwave series from Prolabo. Notwithstanding their performance similarities, there is a substantial difference between FMASE and Soxhlet extraction. Thus, the FMAS extractor is usually equipped with glassware of a design similar to that of the Soxhlet extractor, which can be substituted by a pump serving the functions of the glassware with additional advantages such as the ability to couple the FMAS extractor with other dynamic devices for on-line cleanup and detection. For example the device was coupled to a fluorimeter as shown in Fig. 5.10 in order to monitor the extraction kinetics of fluorescent analytes such as PAHs and


201

Fig. 5.10. Approach to the matrix-independent removal of fluorescent compounds. (A) Controlled FMASE: RV recycling valve. (B) Flow-injection manifold: IV injection valve. (Reproduced with permission of the American Chemical Society.) identify the end of the leaching step irrespective of the sample matrix concerned, thus avoiding exceedingly long extraction times [61]. The main drawback of the FMASE prototype was the difficulty of using water or other high-boiling point solvents as extractants owing to the length of the glassware required for evaporation, condensation and dropping of the solvents on the samples, which considerably slowed the process. In order to enable the use of water as extractant, as well as to improve automatability and operability, a new prototype based on the same principles as the previous FMAS extractor was designed by Luque de Castro et al. and constructed by SEV (Puebla, Mexico). The new prototype, called MIC V (Fig. 5.11), consists of a single unit where the shortened glassware pathway allows reception of the water vapour on a condenser connected to the top of the sample-cartridge vessel with minimal heat losses on the way to its subsequent condensation and dropping on the solid sample [62]. The system operates with two extraction units, which allows the simultaneous processing of two samples. Also, it includes an optical sensor that is positioned at the desired syphon height to have the magnetron start irradiation of the sample when the solvent reaches the preset level. A solenoid valve in the bottom of the syphon is switched on at the end of the irradiation process to empty the sample vessel. The equipment operates similarly to a conventional Soxhlet extractor (i.e. the sample is continuously brought into contact with fresh, recycled solvent), the sole difference being that the sample receives microwave

202


Fig. 5.11. Novel focused microwave-assisted extractor design. (Reproduced with permission of SEV, Mexico.)


203

irradiation over a preset period. This prototype is being tested at present and is expected to be marketed shortly. Microwave-ass&ted drying system A special drying rotor has been used to dry samples using microwaves [4]. The system consists of a cylindrical chamber through which an air stream is circulated after introduction via 6 filter discs located at the cylinder's top plate. This allows the chamber to be steadily purged with clean air, which drives off the moisture released by the sample. Samples, placed in Petri dishes made of borosilicate or quartz glass, rest on a porcelain desiccator plate. Porcelain and the borosilicate glass walls of the cylinder are both slightly self-heating under microwave irradiation, which avoids condensation of moisture even when large amounts of water evaporate (e.g. from sewage sludge samples). Microwave-assisted distiller Microwave energy has also been used to facilitate the distillation of various compounds from solid samples [11,63-65]. Figure 5.12A depicts a typical microwave-assisted distillation apparatus. It consists of a laboratory-made polytetrafluoroethylene (PTFE) vessel of 120 ml inner volume and 10 mm wall thickness, and a 60 ml collector flask. The screw cap of the vessel includes a drilled PTFE disc located on its upper part that avoids sample ejection during distillation. A hole made in the vessel cap provides a means for evacuating volatile compounds via a PTFE tube that is passed through the vent holes of the microwave cavity and is then adapted to a glass line finished by a bubbler which is inserted in the collector flask. A domestic microwave oven equipped with a 2450 MHz magnetron is normally used for this purpose, altered only as required to connect the sample vessel with the collector flask. A more complicated system (Fig. 5.12B) was developed by Conte et al. [66,67] with a view to coupling distillation with a solid-phase absorbent trapping step. The microwave oven (A) was modified as follows: an opening was drilled in the oven for insertion of a probe and another one in its outer casing for introducing a Teflon gas transfer line (I). The Teflon sparge gas tube (E) was inserted through an opening in the bottom of the outer case and then through a small opening in the inner compartment. The flow of helium sparge gas was regulated by a rotometer (H). Tube E was connected to a check valve (G) to prevent reversed flow of liquids during and after distillation. The sparge gas tube (E) was fitted through a Teflon male tee (C) and sealed in place with a Teflon reducing ferrule (F). Component C was connected to a 250 ml thick-wall hydrogenation flask (B) via a Teflon nut (D) furnished with a Teflon O-ring. Argon was used to purge the distillate formed from the microwave radiation through tube I and the flow was directed from I into the condenser (N) via a tee-adaptor (K) and a Teflon union (M). The tee adaptor was attached to I and a Teflon rod (L) by means of a Teflon bushing and O-ring (J). Component L was used as an opening for the addition of the elution solvent. The temperature of the condenser (N) was controlled by a benchtop recirculating cooling unit (S). A support disc (O) was attached to the bottom of the condenser by means of an adaptor (P). The condenser accommodated a stainless steel union having a male Luer lock fitting epoxy-glued to one side (Q). An appropriate solvent material was secured to the Luer fitting.

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205

This design affords shorter analysis times with minimal solvent consumption; also, no preconcentration step is required as preconcentration is achieved simultaneously with distillation by retaining distilled analytes on the solid-phase material. This device features a broad range of uses in the analysis of semi-volatile compounds in solid samples with high moisture contents. Microwave-assisted robotic methods

The high flexibility of a robot station affords the insertion and/or exchange of modules. Such is the case with the system designed by Torres et al. [68], who incorporated a microwave treatment module with a view to developing a method for the determination of trace metals in soils where such steps as sample weighing, reagent and solvent addition, sample digestion and insertion into the measuring equipment would be done in a fully automatic manner. Special robotic stations have also been used for the automatic simultaneous or sequential microwave treatment of several samples. Thus, the system developed by Ciommer et al. consists of a robot hand with special fingers for grasping digestion vessels, a vibrator hand, a pipetting hand, a rotation hand, opening and dosing stations, a sample balance module, a sample rack for 90 samples and 5 digestion vessel racks with 8 positions each. The microwave section was modified so that the digestion vessels and the sealing from the automatic handling section could be accommodated and its operation fully automated [69]. In both cases, the inclusion of the microwave digester in the robotic station dramatically decreased the time required for sample treatment as compared to conventional stirring-based methods, with a concomitant increase in sample throughput. 5.3.4. Closed versus open microwave systems

The earliest microwave systems adapted for laboratory work were of the closed-vessel type; however, many were superseded by open-vessel systems that were developed to circumvent their shortcomings. In any case, each type of system has its own advantages and disadvantages, so neither can be said to be the better choice for all microwave-assisted treatments of solid samples. The advantages of closed-vessel systems can be summarized as follows: (a) They can reach higher temperatures than open-vessel systems because the boiling points of the solvents used are raised by the increased pressure inside the vessel. The higher temperatures in turn decrease the time needed for the microwave treatment. (b) Losses of volatile substances during microwave irradiation are virtually completely avoided by virtue of the absence of vapour losses.

Fig. 5.12. (A) Microwave-assisted distillation set-up- C: collector; TL: tansfer line; V: vessel; WR: water reservoir. (Reproduced with permission of Elsevier.) (B) Microwave-assisted distillation/ solid-phase extraction apparatus. The parts designated by capital letters are described in the text. (Reproduced with permission of the American Chemical Society.)

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(c) Less solvent is required. Because no evaporation occurs, there is no need to continually add solvent to maintain the volume. Also, the risk of contamination is avoided as a result. (d) There is little or no risk of airborne contamination. (e) The fumes produced during an acid microwave digestion are contained within the vessel, so no provision for handling potentially hazardous fumes need be made. On the other hand, the use of closed-vessel systems is subject to several shortcomings, namely: (a) The high pressures used pose safety (explosion) risks derived from the generation of hydrogen in the acid digestion of metals and alloys. (b) The amount of sample that can be processed is limited (usually less than 1 g for inorganic samples and 0.5 g for organic samples). (c) The usual constituent material of the vessel, PTFE, does not afford high solution temperatures. (d) These systems are rarely suitable for organic compounds. (e) The single-step procedure excludes the addition of reagents or solvents during operation. (f) The vessel must be cooled down before it can be opened after the treatment. (g) The use of porous PTFE can result in memory effects. (h) Unlike digestion, leaching involves a mass-transfer equilibrium of the analytes between the sample matrix and the leaching agent that hinders quantitative removal of the target species. Atmospheric pressure (open-vessel) microwave sample preparation can be as effective a method as closed-vessel microwave sample preparation methods or even more so. The use of atmospheric pressure provides substantial advantages over pressurized vessels, namely: (a) Increased safety resulting from operating at atmospheric pressure with open vessels containing, for example, gas-forming species. (b) The ability to add reagents at any time during the treatment, which enables sequential acid attacks in the case of digestion. (c) The ability to use vessels made of various materials including PTFE, glass and quartz. (d) The ability to operate at high temperature with quartz material when using sulphuric acid near its boiling point to destroy organic compounds in digestion methods. (e) The ease with which excess solvent can be removed to ensure complete dryness of the digest or extract. (f) The ability to process large samples. (g) No need for cooling down or depressurization. (h) The low cost of the equipment required. (i) The ability to develop several leaching cycles until quantitative removal of the target species is achieved.


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In addition to these advantages, the following specifications would make open-vessel systems even more useful:

(a) Highly efficient transfer and precise control of the energy deposited into the sample. (b) Fully automatic operation. especially those with a high carbon (c) The digestion of large (3-10 g) samples content - - produces large amounts of gas and vapours. In open vessels, these are released by the reaction mixture and continuously swept from the headspace above the sample. Thus, in contrast to closed vessels, completion of gas-forming reactions is favoured as per Le Chatelier's principle. (d) Open-vessel operation is more suitable with thermolabile species (e.g. organometals) as it uses low temperatures relative to closed-vessel systems. Despite their many advantages, open-vessel systems are also subject to several shortcomings, namely: (a) The ensuing methods are usually less precise than those developed using closedvessel systems. (b) The sample throughput is lower as most open systems cannot process many samples s i m u l t a n e o u s l y - closed-vessel systems can handle 8-14 samples at once. (c) The operation times required to obtain results similar to those of closed-vessel systems are usually longer. (d) Digestion is especially cumbersome with some samples owing to the difficulty of reaching the drastic conditions they require.

5.4. VARIABLES GOVERNING MICROWAVE-ASSISTED PROCESSES Performance in microwave-assisted processes depends on a number of variables including the microwave power output, exposure time, solvent and sample size used. However, specific treatments such as digestion are additionally dependent upon factors such as the digesting acid, pressure and its relationship to temperature in closed-vessel systems, the residence time in flow systems, the number of cycles used in an FMAS extractor, etc., all of which should be optimized for each specific situation. 5.4.1. Microwave power and exposure time

The principal variables to be considered in developing a microwave-assisted analytical method are the applied power and the irradiation (exposure) time. The power and the corresponding time will depend on the type of sample and reagent used. Usually, these two variables have opposing effects: for a given process, the use of a high microwave power affords a decreased exposure time; on the other hand, the use of a low power entails irradiating the sample for a longer time to apply the same amount of energy. In theory, one should use a high microwave power to reduce the exposure time as much as possible. This is the rule to be followed in most digestion processes, i.e. to use

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the highest power afforded by the microwave equipment in order to minimize the digestion time [2]. Occasionally, however, using a very high power is inadvisable; such is the case with digestion processes involving acid mixtures, abrupt heating of which can cause them to react explosively (e.g. the sulphuric-nitric acid mixture in the presence of organic material) or in extraction processes where complete dissolution of the sample should be avoided in order to minimize the effect of potential interferences. In some cases, using a very high microwave power decreases the extraction efficiency through degradation of the sample or its analytes [59,60], or rapid boiling of the solvent in open-vessel systems (which hinders contact with the sample). In the extraction of herbicides from acid soils using an open-vessel focused microwave system, the application of microwaves over short, intermittent periods resulted in substantially increased extraction efficiency relative to the continuous application of microwaves, which caused the solvent to boil and hindered sample--extractant contact [43]. In dynamic digestion systems where the sample is inserted as a slurry in a Teflon coil, the microwave exposure time is directly related to the residence time of the slurry inside the oven, which in turn depends on the length of the digestion coil and the flow-rate of slurry through the coil. With focused microwave-assisted Soxhlet extraction, the process develops in cycles where the sample is brought into contact with volumes of fresh extractant, thereby favouring displacement of the analytes from the matrix to the extractant. The sample is irradiated with microwaves for a preset time during each cycle. The number of cycles and the irradiation time used in each cycle are thus two variables to be optimized in using a focused microwave-assisted extractor. 5.4.2. Temperature and pressure

Temperature is a key variable in most analytical processes. In microwave-assisted processes, it plays a prominent role and affects the rate of some reactions, the degradation of thermolabile species and the solubilization of some substances, among others. A number of devices have been developed for monitoring or even controlling the temperature, some of which are commented on in Section 5.3. Pressure is a highly influential factor in closed-vessel systems. The development of vessels capable of withstanding pressures as high as 41 bars has enabled digestion at very high temperatures with pure or mixed liquid acids, thereby dramatically increasing the digestion efficiency and decreasing the exposure time (see Section 5.5). Some devices also discussed in Section 5.3 allow the pressure to be both monitored and controlled. 5.4.3. Type of solvent The choice of solvent in a microwave-assisted sample treatment is a direct function of the type of treatment used. Thus, digestion and hydrolysis are best done with aqueous solutions of pure or mixed acids, whereas selective extraction of organic compounds from a sample matrix usually requires an organic solvent. The microwave-assisted extraction of non-polar compounds usually entails the adoption of a compromise since, although these compounds are more readily dissolved in


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non-polar solvents such as n-hexane, the interaction of microwaves with the solvent depends on its dielectric constant (the greater e, the stronger the interaction); this requires the use of a mixture of solvents of different polarity in many cases. For example, the most suitable solvent for the microwave-assisted extraction of organic compounds such as PAHs and PCBs is a mixture of hexane and acetone. In specific cases such as the extraction of organometal compounds, which is discussed in greater detail in Section 5.5, the use of methanol acidified with acetic acid allows organometals to be extracted in a rapid, efficient manner with no degradation. The solvents of choice for digestion (microwave-assisted digestion included) are solutions of both oxidizing (e.g. HNO 3, H2SO4) and non-oxidizing inorganic acids (e.g. HCI, HF). The acid or mixture of acids should be chosen for its efficiency in decomposing the matrix. Thus, knowledge of the sample matrix and its major elements and compounds is essential with a view to selecting the appropriate acid to ensure complete sample dissolution. Combinations of acids are frequently used to digest a sample matrix. For example, hydrofluoric acid alone is inappropriate for the decomposition of plant material. However, if a siliceous component is present, hydrofluoric acid is added to nitric acid in order to release trace elements that would otherwise remain trapped with the silica. These combinations must be chosen on the basis of the chemistry of the sample matrix [70,71]. The high significance of microwave-assisted digestion and its wide application warrants some comments on the characteristics of the different acids usually employed in the process and also of hydrogen peroxide, not an acid itself but widely used for digestions on account of its oxidizing power. Nitric acid

Nitric acid is the primary choice here on several grounds. It is an oxidizing acid that can dissolve most metals to form soluble metal nitrates. It has poor oxidizing strength below 2 M but is a powerful oxidizing acid in concentrated form. Its oxidizing power can be enhanced by addition of chlorate, permanganate, hydrogen peroxide or bromine, as well as by increasing its temperature and pressure. Most metals and alloys are oxidized by nitric acid; however, gold and platinum are not oxidized and some metals are passivated when attacked by concentrated nitric acid. Such metals can be dissolved by using a combination of acids or a dilute nitric solution. At temperatures in the region of 180~ nitric acid can decompose most organic molecules, converting hydrocarbons into carbon dioxide and water. Hydrochloric acid

Hydrochloric acid is a non-oxidizing acid that exhibits weak reducing properties during digestion. Concentrated HC1 is an excellent solvent for some metal oxides and for metals that are oxidized more easily than hydrogen. At a high pressure and temperature, many silicates and numerous other refractory oxides, sulphates and fluorides are attacked by HC1 to produce generally soluble, chloride salts.

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Because it is not an oxidizing agent, concentrated hydrochloric acid is normally not used to digest organic materials. Nevertheless, it is an effective solvent for basic compounds such as amines and alkaloids in aqueous solutions, as well as for some organometallic compounds. The hydrolysis of natural products with HCI is a routine preliminary procedure for the analysis of amino acids and carbohydrates. Hydrofluoric acid Hydrofluoric acid is a non-oxidizing acid the reactivity of which is based on its strong complexing capacity. It is most commonly used in inorganic analysis because it is one of the few acids that can dissolve silicates. Its strong complexation capabilities prevent the formation of sparingly soluble products of various metals and increases their solubility and stability. Digestion with HF produces mainly soluble f l u o r i d e s - by exception, the fluorides of alkaline earths, lanthanides and actinides are insoluble or sparingly soluble at most. In order to improve digestions, HF is routinely combined with another acid such as HNO 3. Insoluble fluorides may frequently be redissolved by removing the HF after digestion. Phosphoric acid Hot phosphoric acid has been successfully used to digest ion-based alloys when HCI would have volatilized specific trace constituents. Phosphoric acid can also digest a wide range of aluminium slags, iron ores, chromium and alkali metals. The temperature and pressure profiles for H3PO4 indicate that temperatures of 240~ can be attained with just 3 atm. Because of its low vapour pressure, relatively high temperatures can be reached without disrupting the digestion. Sulphuric acid While dilute sulphuric acid exhibits no oxidizing properties, in concentrated form the acid is capable of oxidizing many substances. In fact, concentrated H2SO4 is an effective solvent for a wide range of organic tissues, inorganic oxides, hydroxides, alloys, metals and ores. However, concentrated sulphuric acid (98.7%) has a boiling point of 339~ which exceeds the working range of all Teflons. As a result, quartz is the material of choice for the vessels used in H2SO4 digestions. Sulphuric acid is commonly used with other acids and reagents. Two of the most frequent combinations are with perchloric acid or hydrogen peroxide. Sulphuric acid can act as a dehydrating agent and dramatically increase the oxidizing power of perchloric acid; however, the mixture can react explosively with organic matrices in closed vessels or if heated too rapidly. Perchloric acid Hot HC104 is a strong oxidant which attacks metals that are unresponsive to other acids. Perchloric acid also effects the thorough decomposition of organic materials.


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Because of its oxidizing capacity, the hot acid is frequently used to take elements to their highest oxidation state. Cold concentrated and hot diluted H~PO4 pose little hazard; however, too concentrated HC104 is potentially explosive when in contact with organic materials and easily oxidized inorganics. Extreme safety precautions are required when using this concentrated acid at high temperatures. Because of this potential hazard, expensive acid hoods, special scrubbers and duct work are needed to handle this acid.

Hydrogen peroxide Typically, concentrations of about 30% hydrogen peroxide are used for digestion; more recently, however, 50% solutions have become available. Hydrogen peroxide alone can react explosively with many organics, especially in the more concentrated form. Hydrogen peroxide is usually combined with an acid because its oxidizing power increases as the acidity increases. The combination of H202 and sulphuric acid forms monoperoxosulphuric acid (H2SO~), a very strong oxidant. Because of its oxidizing power, hydrogen peroxide is frequently added after the primary acid has completed a pre-digestion of the matrix. The hydrogen peroxide can complete digestion and the potential safety hazards are minimized. In this respect, H202 is used similarly to HCIQ. 5.4.4. Influence of sample viscosity and sample size on microwave heating The viscosity of a sample reflects its ability to absorb microwave energy because it affects molecular rotation. The effect of viscosity is best illustrated by considering ice water. When water is frozen, the water molecules become locked in a crystal lattice. This greatly restricts molecular mobility and makes it difficult for the molecules to align with the microwave field. Thus, the dielectric dissipation factor of ice is low (2.7 • 10 4 at 2450 MHz). When the temperature of the water is increased to 27~ the viscosity decreases and the dissipation factor rises to a much higher value (12.2). The input microwave frequency also affects the penetration depth of the microwave energy. As noted in Section 5.2.1, at a given input frequency, the greater the dissipation factor of a sample is, the less it will be penetrated by microwave energy. In large samples with high dissipation factors, the heating that occurs beyond the penetration depth of the microwave energy is due to thermal conductance through molecular collisions. Therefore, temperatures at or near the surface will be higher. Because boiling and other agitation increase the rate of thermal conductance, surface heating is not a problem unless penetration is very low and sample heating extremely superficial. In such a case, heat losses through the vessel walls can be significant and increase the sample heating time. Although the small sample size used in most analytical dissolution processes has some advantages, it is also subject to a least one disadvantage: the amount of microwave energy absorbed decreases with decreasing sample size. With small sample sizes, a substantial amount of energy is not absorbed but reflected. Reflected energy can damage the magnetron, so, in using small samples for analytical work, it is advisable to employ microwave systems designed to protect the magnetron from reflected power.

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5.5. APPLICATIONS OF MICROWAVES TO SOLID SAMPLE TREATMENT Microwave-assisted sample pretreatment is being increasingly used in analytical laboratories all over the world. This section describes the principal applications developed since the inception of microwave devices for solid sample treatment in the analytical laboratory. Owing to the large number of papers published on the topic since then (in excess of 2000), only the most salient aspects of the body of information they contain are discussed here in order to provide readers with a global picture of the potential of this auxiliary energy for treating solid samples. Readers wishing to go deeper into any specific aspects are thus referred to the corresponding publications. For consistency with the subject matter of this book, applications of microwaves to liquid samples, microwaveassisted elution [10], thermospraying [72], ozonization [73] and desolvation [74-76] are not dealt with here. Microwave-assisted digestion and extraction are discussed extensively as they constitute the two most relevant areas and those producing the largest numbers of analytical applications of microwaves. Digestion applications are classified according to the type of matrix involved as, in most cases, digestion is followed by the determination of metal species, so the conditions that govern the digestion efficiency depend on the type of matrix concerned. Microwave-assisted Kjeldahl methods are also commented on, at the end of this section, because of the good results they have provided to date. On the other hand, extraction applications are classified according to the analytes involved as the efficiency of the extraction step is dictated by the particular species to be leached. Finally, other salient uses of microwaves for treating solid samples such as microwaveassisted drying, distillation and protein hydrolysis are also briefly described.

5.5.1. Microwave-assisted digestion Sample digestion methods must efficiently decompose the sample matrix so that the target analytes can be thoroughly released from it and solubilized in a form compatible with the determination method of choice. Ever since Abu Samra reported in 1975 the earliest use of microwaves as a heat source for wet digestion [1], microwave digestion has gradually gained widespread acceptance as an effective means of sample treatment. With the aid of microwaves, not only have digestion times been dramatically reduced (by a factor of 2-5), but also other benefits such as reduced contamination and reagent and sample usage, and improved safety, have been derived. The advantages of microwave-assisted digestion have led to its wide application as an effective sample pretreatment method, mainly for the determination of metals in a wide range of sample matrices; in fact, many of the ensuing methods have been adopted as references by several bodies [77-81]. An increasing number of laboratories are replacing conventional methods with others based on this technology, as reflected in the ever increasing amount of material published on the subject [2,82]. The reports on solid sample digestion involved geological, biological (zoological, botanical, food), soil, sediment and sludge samples, coal and ash, metals and synthetic materials.


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As stated in Section 5.3, there are two main types of microwave units, namely: closedvessel systems, which use pressurized containers, and open-vessel systems, which use focused microwaves at atmospheric pressure. Both types of system have been used to digest solid samples and each has its own advantages and disadvantages, so it is impossible to recommend either as the more suitable overall.

Geological samples Geological materials such as ores, rocks and minerals often contain immutable matrices resistant to acid attack. Many such matrices consist of refractory minerals with high melting points or other compounds that may require several hours of heating in acids, or fusion processes, for successful digestion. The introduction of microwave-assisted digestion has been a major breakthrough in geochemical analysis, as shown by the many studies involving the use of microwave energy for the digestion of geological samples [83-93]. Regarding microwave equipment, most workers have used modified domestic ovens [84,89,91,94,95] for digestion. The most common modification is the incorporation of a device for purging the oven cavity of fumes to a hood using either compressed air, CO 2 gas or fan-forced air. Most microwave-assisted digestions have been performed using sealed vessels and a pressure release mechanism [85,96] plus pressure and/or temperature monitors [96-98] in some cases. Some special systems have been designed for specific purposes [96-98] or for safer use of closed vessels and microwave energy [84,89]. Also, some workers have placed closed vessels inside containers in order to prevent leaked fumes from destroying the oven cavity [84,86,89,91,96]. Validation of microwave-assisted digestion methods has relied on the use of common certified reference materials and comparisons have also been made with other, conventional procedures such as prolonged heating with acids with a view to testing the reliability, accuracy and precision of microwave-based methods [83,85,88,92,93,96, 99-105]. By judicious adjustment of the different variables involved (e.g. the choice of acid mixture, power setting and exposure time), it has been found possible to digest most types of geological samples using microwave-assisted digestion. Precision and accuracy are generally as good as or even better than those obtained with conventional digestion methods [82]. Simple nitric or hydrochloric acid digestions suffice for the determination of some elements. For example, good Fe recoveries from iron ore samples were obtained using a simple hydrochloric acid digestion; by contrast, limestone rocks required the additional use of hydrofluoric acid [106]. Other acid mixtures such as HNO~-HC1 [97,98,102], aqua regia-HCl [90,96,105] and HNO3-HC104 [86,95,107] have also been used for the efficient digestion of geological samples.

Biological samples The usual aim of digesting biological samples is the subsequent determination of metal traces potentially acting as nutrients or toxins, or even causing diseases such

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as urolithiasis [108]. One of the major problems in determining trace metals in biological (zoological, botanic, food) samples arises from the need to dissolve organic matter [109]. There are many reports on the use of microwave digestion with biological samples [110-142]. The high pressure generated from the use of closed vessels raises the boiling point of the acid(s) employed and substantially increases the dissolution rate of these organic matrices. For example, nitric acid normally boils at 120~ however, in a closed vessel at 5 atm, it boils at 176~ [14]. This is 56~ above its normal boiling point and increases its oxidation potential and accelerates its reactions. Under classical hot-plate conditions, the organic matrices of biological samples would remain intact due to the low boiling point of nitric acid. Under these circumstances, reagents such as perchloric acid would have to be used to ensure complete destruction of the organic matter. The closed-vessel microwave approach dispenses with the use of perchloric acid, which is potentially explosive upon contact with readily oxidized inorganic or organic materials, especially at high temperatures. Great care should be exercised when heating perchloric acid under pressure, even by itself: it has been shown that closed vessels heated with only 5 ml of HCIO 4 remain pressurized even after cooling with liquid nitrogen. Instead of vaporizing and condensing like other acids, perchloric acid at high temperatures generates chlorine gas via an irreversible decomposition reaction. The chlorine gas is not easily frozen and maintains a high pressure within the vessel. Despite the hazard, some authors have used perchloric acid mixtures under pressure to decompose biological samples [33,143-148]. Fortunately, pressure digestion involving other acid mixtures can be used with acceptable results [149]. However, even when other acids are used, the substantial change in internal pressure generated by the production of CO 2 and NO 2 as organic matrices are decomposed must be anticipated and controlled to prevent vessel rupture, oven damage and possible personal injury. As a safety precaution, vessels with some kind of pressure release mechanism are recommended. Biological (zoological, botanical, food) samples are primarily composed of three basic constituents, namely: carbohydrates, proteins and lipids. Carbohydrates are the first constituents to decompose, at approximately 140~ followed by proteins at ca. 150~ and lipids at about 160~ [14,150]. At each of these temperatures, significant increases in pressure are generated in the vessel with virtually no increase in temperature. Thus, for safety reasons and for determining the most efficient decomposition temperature, the analyst should have some knowledge of the constituents of the sample matrix. Although biological samples are most often digested in closed-vessel systems, focused microwaves have also been used for this purpose in some instances [151-155]. However, the use of microwave-transparent, inert, closed vessels appears to be the method of choice for destroying organic matter [156]. The loss of analytes through volatilization or adsorption onto the vessel walls is negligible despite the high pressures and temperatures associated with the digestion. In most instances, such high pressures and temperatures result in complete dissolution of the organic matter that often remains after digestion with conventional techniques [82]. Much of the research on biological sample digestion has included results from standard reference materials for validation purposes. Also, much of the development work has been conducted in parallel with a more traditional method such as conventional heating [157,158] and also with new methods such as pressurized ashing [117].


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The results provided by microwave digestion have been found to be comparable or even better in most cases, with advantages such as shorter operational times, higher safety concerning the use of acids and higher precision as a result of the use of automated equipment [ 158]. There are some contradictions as regards the efficiency of the different acids used for digestion in the determination of the same element. For example, a simple nitric acid digestion was successfully used for the determination of iron in cocoa [ 126], horse kidney [127], total diet [117], bovine liver [128,133,139], mussels [128], chlorella [128], sargasso [114] and pepperbush [114] samples. However, Mingorance et al. [132] obtained low, imprecise results using a similar method with botanical samples. A slight improvement was obtained with nitric acid and hydrofluoric acid digestion, but the results were still outside the certified range. Whether digestion with hydrofluoric acid is necessary for the determination of aluminium is a controversial subject. Thus, low aluminium recoveries from citrus leaves and pine needles were obtained using closed-vessel HNO3-HF digestion [129] that were improved by employing both acids in combination with hydrogen peroxide. Low recoveries were also obtained from spruce needles after HNO3-H202 digestion [123], as well as from shellfish [39], total diet samples [140], apples and peach leaves [135] following closed-vessel digestion with nitric acid alone. On the other hand, the addition of hydrofluoric acid to the HNO3-H202 mixture provided improved results. However, evidence also exists which suggests that digestion with hydrofluoric acid is unnecessary for some samples. For example, good results have been obtained for aluminium in total diet [117] and citrus leaves [142] after a simple nitric acid digestion. A combination of nitric acid and hydrogen peroxide was successfully used for the digestion of wheat flour, but the results for rice flour where slightly high [134]; recoveries were not increased, however, by including hydrofluoric acid in the procedure. Similar inconsistencies have been encountered in the determination of other elements such as selenium [ 125,133], arsenic [ 118,130], copper [ 119,123] and zinc [ 120,122]. On the other hand, methods for the determination of mercury are slightly more consistent, with many workers using a closed-vessel nitric acid digestion procedure. Good results have thus been obtained with pig kidney [124,141], mussel tissue [138], cod muscle [137], citrus leaves [136], pine needles [136], albacore tuna [131] and fish tissue [124], among others. There are various approaches to the on-line digestion of biological samples. The development of these flow-through digestion systems for coupling to a variety of instruments is a further step towards complete automation. Virtually all the designs described in Section 5.3, whether of the closed-vessel [32,33] or open-vessel type [33,37,39,46-48, 50,146,159-161], have been used for the on-line digestion of biological samples - - particularly open-vessel systems where the sample is inserted as a stabilized slurry. This has probably been the result of the ease with which these systems can be coupled to a detector (especially an atomic detector) and of their low cost and complexity in relation to highpressure systems for on-line microwave-assisted digestion. The greatest advantage of on-line digestion methods over batch methods is that they minimize the problem of acid fumes produced during the digestion of biological samples in the traditional Teflon vessels used in batch treatments. Gas evolution in the on-line methods, however, can lead to problems resulting in pressure build-up and disturbances

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of the carrier flow. In some cases, air carrier streams have been used in order to sweep the gases formed during digestion [48]. Notwithstanding the above-described advantages of on-line open-vessel systems and their more extensive development, high-pressure on-line systems such as that of Fig. 5.5A are especially effective for digesting biological samples, which requires strong, fast oxidation of the organic matter. The main advantage of these high-pressure on-line systems is the substantially increased digestion temperature achieved, which significantly raises the oxidation potential of acids such as HNO 3. This enables the digestion of tough organic compounds and accelerates the oxidation reaction in general, thus shortening the digestion time [33].

Sediment, soil and sludge samples Soil and sediments are highly variable, complex matrices; the latter are considered to be the ultimate sink of heavy metals released into the environment [162] and also reservoirs for other environmental pollutants such as organochlorine insecticides [163]. The relative ease with which metals are released from, or bound to, soils and sediments is poorly understood. With increasingly stringent environmental and health regulations, the demand for environmental analysis and the range of sample types have grown enormously over the past decade. The most important type of sludge is that resulting from the treatment of sewage. Although there are appreciable benefits in the application of this sludge to farmland [164,165], there is also the risk of toxic elements and bacteria accumulating in the soil if the sewage is not properly treated. The accumulation can lead to contamination of the soil, ground water, lakes and streams, and eventually of the food chain (e.g. fish, crops and livestock), so the analysis of the metal content in sludge is also mandatory for environmental and health regulations. Microwave-assisted digestion is gaining wide acceptance as the method of choice for the pretreatment of these types of samples [93,162,164, 166-185]. In fact, most official microwave-assisted digestion methods are used prior to the determination of metal species in soil, sediments and sludges [77-81], and employ nitric acid, alone or in mixture with hydrochloric or hydrofluoric acid, as the main reagent. Most on-line microwave-assisted digestions of these types of samples have been done by introducing them as acid slurries and coupling the manifold to a suitable atomic detector [24,49,51,52,54].

Metallic samples The determination of metal purity and the elemental composition of alloys is of utmost importance to the metallurgical industry. Microwave-assisted digestion is often well-suited to metals and metallurgical samples that pose no difficulty and dissolve readily and safely with the aid of microwaves [148,186-196]. For example, hydrofluoric acid can be used in closed vessels to digest silicate matrices and stop the hydrolysis of refractory elements without loss of volatile fluorides or passivation. After cooling, boric acid can be added to complex unreacted hydrofluoric acid [14]. The solid sample itself may absorb microwave radiation, thus creating a heated surface on which the acid or acids can react. Microwave muffle furnaces are commercially available [197] based on oven linings made


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out of the highly efficient microwave absorber silicon carbide. This microwave absorber rapidly heats up the oven cavity to ashing or fusion temperatures. Other solid samples may reflect microwaves and create high sample/acid interface microwave densities. This can result in very fast heating of the acid(s) used and create turbulence and sample agitation, both of which sweep sample surfaces clean of acid-resistant coatings and expose fresh, unexposed sample surfaces to acid attack. The use of microwave energy in the presence of metallic materials is not recommended in all circumstances. Specifically, the interaction of some metals with microwave energy can result in uncontrolled sparking and electrical arcing, possibly causing damage to electrical components and the magnetron. This can be a problem and is a safety hazard when performing closed-vessel digestion. Some metallic materials (especially iron-based alloys) spark when subjected to microwave energy. Such sparking can ignite the hydrogen gas generated in the digestion of metals [14,194]. Miscellaneous samples

Microwave-assisted digestion has been applied to other types of samples such as ceramics [198,199], air particulate matter [200-202], polymers [203,204], lithographic materials [205], coal and ash [206], among others. As with the previous samples, the main purpose was the determination of their metal contents. The results provided by microwave-assisted digestion are similar to those obtained with conventional methods. Microwave-assisted Kjeldahl methods

Monitoring total nitrogen in soil, which is of special interest to those concerned with plant growth, has traditionally been a cumbersome procedure. The most common method for extracting total nitrogen is Kjeldahl digestion, a procedure developed by Johan Kjeldahl in 1882 and reviewed by McKenzie in 1994 [207]. A Kjeldahl digestion simply involves boiling the sample in sulphuric acid for several hours in the presence of a mercury or copper catalyst. This extraction yields the nitrogen as ammonium sulphate. The time required for the extraction of nitrogen from samples in a conventional Kjeldahl step ranges from 2 to 4 h, which makes it the rate-determining step of the overall analysis. When a large number of samples are to be analysed, this time requirement can prove very costly and an enormous drain on resources and manpower. In 1988, Alvarado et al. [208] investigated the use of closed test-tube microwave Kjeldahl digestion in a domestic microwave oven for the determination of the total nitrogen content in various foods. Some very encouraging results were obtained in the form of improved precision and reduced digestion times. For example, the digestion of cassada leaves took 130 min with the conventional method and 14.6 min with the aid of microwaves. The method did, however, require continuous attention from the operator, turning the microwave power on and off so as not to allow the pressure to exceed the pressure that the tubes can tolerate. Later developments, again in investigating the digestion of food, were reported by Feinberg et al. [209], with the introduction of a more sophisticated open-vessel focused microwave digestion system. They reported that using a metal catalyst was unnecessary if hydrogen peroxide was employed. This procedure, at

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around 40 min, was still quite lengthy and provided little advantage over thermal methods. In 1996, Collins et al. [210] used a focused microwave system and achieved similar results. However, the system still only had the ability to handle one sample at a time. More recently, Mason et al. [211] reported a method that combines the microwave Kjeldahl system and a flow-injection manifold with microwave-enhanced colour development; the new system greatly reduces the time needed to determine total nitrogen and increases throughput from 36 samples/h on the thermally heated system to 60 samples/h on the microwave-heated system [211 ]. 5.5.2. Microwave-assisted extraction

Microwave-assisted extraction (MAE) is the process by which microwave energy is used to heat solvents in contact with solid samples and to partition compounds of interest from the sample into the solvent. Extractions are done in closed or open microwavetransparent vessels where the solvent and sample are combined and then uniformly exposed to microwave energy. Partitioning may occur by any one or several of the following three heating mechanisms: (a) a single solvent or mixture of solvents possessing high dielectric loss coefficients; (b) solvent mixtures of high and low dielectric loss; (c) susceptible samples with a high dielectric loss in a solvent of a low dielectric loss. Although the traditional Soxhlet and solvent extraction techniques are widely accepted, they have inherent limitations and problems. Thus, Soxhlet extraction requires 12-24 h in most cases and uses high volumes of organic solvents (hundreds of millilitres). In contrast to conventional methods, microwave-assisted extraction can reduce the extraction time to less than 30 min and solvent consumption to under 50 ml [12]. Moreover, the recoveries obtained with microwave-assisted extraction are comparable with those provided by alternative extraction methods [7]. Unlike digestion, microwave-assisted extraction is used mainly with soil and sediments rather than with biological samples. As regards analytes, microwave-assisted extraction is applied primarily to organic pollutants such as PAHs, PCBs and pesticides rather than to metal species. The use of MAE is a continuously expanding area of research at present. As a result, it is difficult to provide a completely updated overview. Rather, selected groups of compounds of interest that have been subjected to MAE are discussed here. Unless otherwise stated, the microwave systems used in these applications were of the open-vessel type, which require none of the drastic pressure and temperature conditions employed with closed-vessel systems. Pesticides

The increasing risks to human health posed by the widespread use of pesticides in our environment is well established [212,213]. Thus, the determination of pesticides in water, plants, soil, sediments, foodstuff, etc., is of major concern today [214,215]. Microwave-assisted extraction provides an efficient, reproducible alternative to classical methods based on Soxhlet extraction or sonication for the extraction of pesticides from environmental samples.


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A wide range of pesticides including organochlorine [216,217] and organophosphorus compounds [218], triazines [219-222], herbicides [43,62] and imidazolinones [223] have been extracted with the aid of microwaves, with recoveries close to 100% in most cases. Sample ageing is an important factor in evaluating a new extraction method as it allows some degree of analyte-matrix interaction to occur and, therefore, provides a more realistic environmental situation. This consideration is very important in pesticide analysis, so ageing studies of the samples to be tested must be performed, as shown by the study on organochlorine pesticides listed in EPA Method 8081. The compounds in question included hexachlorocyclohexane (HCH) isomers, DDT and DDD analogues, as well as endrin and dieldrin [224]. Sand was found to be the cleanest matrix (i.e. it provided the highest recoveries and precision). Co-extractable materials from the topsoil and composted materials are believed to interfere with the analysis and lead to high recoveries of endrin and other compounds. When 45 organochlorine pesticides listed in EPA Method 8081 were freshly spiked onto a series of soils that were aged for a variable time, 38 were recovered to an extent of 80-120%. Most of the compounds were recovered at similar levels after ageing for 24 h - - by exception, captafol, captan and dichlone were poorly recovered. Recoveries also decreased with increased ageing time, which can be ascribed in part to microbial degradation. In any case, they were higher than those obtained for comparable Soxhlet-extracted or sonicated samples by about 7% [225]. In another study, 47 organophosphorus pesticides listed in EPA Method 141A were extracted from freshly spiked and variously aged soil, 35 of them being recovered virtually quantitatively. A few including dichlorvos and fensulfathion were recovered at 50-80%. Spiked samples aged for 14 days fared better: 37 compounds were recovered at > 80%, a few at 50% and some at > 120%. Recovery of poorly extracted compounds from freshly spiked soils always improved in briefly aged topsoils. Continued ageing reversed the overall trend because recoveries of the same analytes decreased after 21 days. This anomalous behaviour was attributed to microbial digestion [226]. Because pesticides persist in the environment and are lipophilic, they accumulate along the food chain, so food, zoological tissue and plant analyses for them are of paramount importance. Monitoring programmes tracking this activity have recently reported the use of MAE to improve sample pretreatment activities required in connection with pesticide analysis and assessment of accumulations. Recoveries of organochlorine pesticides higher than 90% were obtained with 6 samples within less than 40 min for grey seal rubber and spiked pork fat [227], and close to 100% within 20 min for medicinal plants [228]. Recoveries of atrazine and organophosphorus pesticides from oranges ranged from 93% to 101~ with advantages over traditional shake-out extraction methods such as a short extraction time (20 min versus 1.5-2 h), low solvent consumption (10 ml versus 150-300 ml) and automatability for routine sample treatment [218]. Polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) constitute an extensive group of compounds distributed in the environment as a result of natural processes or anthropogenic inputs [229,232]. Traditionally, the extraction of such compounds from solid samples prior to

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analysis has been conducted using the Soxhlet method, which, while being efficient, usually takes more than 6 h and uses large volumes of solvents. Microwave-assisted extraction is an advantageous alternative to the conventional extraction of these compounds [53,233,234]. Several reports indicate that PAHs can be extracted from solid matrices such as soil, plants and animal tissues relatively easily by using simple microwave-assisted extraction [235]. The most effective solution used for leaching PAHs is the acetone-hexane mixture, usually in a 1:1 ratio. Barnabas et al. [236] investigated the effect of varying this ratio on the recovery of PAHs from a native contaminated soil. The combinations ranged from 80:20 to 0:100 hexane-acetone in steps of 10. It was found that, as the solvent mixture became more polar (viz. at a hexane-to-acetone ratio of 10:90 or higher), it was possible to recover greater amounts of PAHs from the native soil. The use of water as extractant was also tested with a view to developing clean methods using no organic solvent. Because of the high hydrophobicity of PAHs, however, the recoveries were very low in spite of the strong interaction between the microwaves and the water. One way of using water effectively for the microwave-assisted extraction of non-polar compounds (PAHs included) is by micelle formation. The ensuing methods provide recoveries close to 100% [3,237,238]. Most studies about the microwave-assisted extraction of PAHs from solid samples have been conducted using closed-vessel systems [12,214,226,236,239-246] and only a few with open-vessel focused microwave devices [57,247-252]. Because open-vessel systems operate at atmospheric pressure, the extraction vessel can be used as a reactor in order to perform on-line purification pretreatments of the total extracts (reagents can be readily added to the medium) [53] or directly introduce the extract into the determination instrument, as in the focused microwave-assisted extractor with on-line fluorescent monitoring of Fig. 5.10, which provides a matrix-independent approach to the extraction of PAHs [611. Polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) were produced from 1930 to 1983 in the form of complex mixtures for a variety of uses (e.g. dielectric fluids in transformers and capacitors, and plasticizers in paint and rubber sealants). Because of their physical and chemical stability, and also of their lipophilic affinity, they are highly persistent and tend to accumulate in sediments and biota in the aquatic environment [253]. Microwave-assisted extraction (MAE) has become a major choice for the extraction of PCBs from solid matrices. In fact, this technique has been used to extract PCBs from a wide range of samples including soil, sediments and animal tissues [254-257], as well as certified reference materials (CRMs) [258]. Normally, the extractant used is the same as that employed with PAHs, viz. a 1:1 hexane-acetone mixture [246]; however, organized media [256] have also provided results similar to those obtained with conventional methodologies such as Soxhlet extraction [259]. For example, the results for a range of Aroclors (1254, 1260, 1016 and 1248) were quite consistent with their certified values. The microwave-assisted extraction of PCB Aroclors 1248, 1254 and 1260 followed


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by enzyme-linked immunosorbent assay (ELISA) was shown by L6pez-/kvila et al. [225] to compare favourably with traditional extraction methods. No degradation of the PCBs was found to occur during heating of solvent-soil suspensions with microwave energy [224,225]. Organometallic compounds Synthetic compounds combining the special properties of metals with a hydrocarbon moiety as a vehicle for their introduction into chemical formulations such as arsones and selenones, arsines, organotins and organomercuries have become widespread in the environment since the early 1960s. For example, roxarsone, a disubstituted phenylarsonic acid, has been used as a growth promoter in animal food production. Triphenyl tin (TPT) and tributyl tin (TBT) are added to marine antifouling paints as growth inhibitors. Recently, several MAE methods were developed for the extraction of organometallic compounds such as 3-nitro-4-hydroxy-phenylarsonic acid [260] and methylmercury [261-263] from sediments and animal tissues. The MAE recoveries were consistently higher than those achieved with sonication or Soxhlet extraction. All species recoveries were matrix-dependent, as previously found with other pollutants, but using methanol acidified with acetic acid under the optimal MAE conditions afforded rapid, efficient extraction of all analytes. Microwave-assisted extraction has also been used as a solid sample treatment prior to speciation analysis [264-266], leaving the organometallic compound moiety intact. This is a prerequisite for a successful extraction procedure to be applied prior to speciation analysis and can be met by careful optimization of the conditions of the microwave attack. Open-vessel treatment is preferred to pressurized bomb systems commonly used in the analysis for total metals because it offers milder reaction conditions - - the increase in temperature is governed to a great extent by the boiling point of the s o l v e n t - and easier control of process variables [266]. Miscellaneous organic compounds Other organic compounds such as phenols [40], hydrocarbons [267], polymer additives [268,269] and natural compounds [270] have also been extracted from a wide range of solid matrices with good results. This confirms that, under optimal conditions of solvent, microwave power and exposure time, etc., the MAE technique is suitable for the extraction of organic compounds from solid samples. One of the most interesting fields of application of microwave-assisted leaching of organic compounds is probably food analysis for lipids. This step, traditionally developed by conventional Soxhlet extraction, has recently been performed with the focused microwave-assisted Soxhlet prototype of Fig. 5.9B. The extractions of oil from olives [58], and sunflower seeds and soybean [271]; those of the lipid fraction of dairy products (milk [272] and cheese [273]); and those of fatty acids from precooked [59] and sausage foods [60] have significant advantages over conventional methods including dramatically reduced extraction times, lower degradation of thermolabile analytes and acceleration of

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other analytical steps such as hydrolysis in milk samples, in addition to completeness of analyte extractions which is not always achieved with conventional methods. Metals

The extraction of metals from solid samples has also been assisted by microwaves [68,274-277], most often with a view to speciation analysis [44,278-282]. Metals such as Fe, Cu, Zn, Pb, Hg, Se, As, Co, Cd, Cr and Ni have been extracted using an acid solution (usually HNO3) in a focused open-vessel system for periods ranging from 3 min for soil samples [68] to 50 min for coal (where metals are much more strongly retained) [46]; the system of Fig. 5.8A allowed the sample to be brought into contact with fresh solvent in four cycles, which resulted in the relatively short extraction time achieved. One clear example of the dramatically reduced extraction times afforded by microwaves is the extraction of Pb, Zn and Cu from calcareous soil [277], where the sequential procedure used proved considerably faster than conventional extraction (2 h versus 20 h). Microwave-assisted extraction has been used both as a pretreatment for chemical speciation (mainly to discriminate As [278,282] and Se species [44,279]) and as a means for accelerating the fractionation of metals in sewage sludge for operational and/or functional speciation [280,281]. The former type of application has usually been developed using open-vessel systems coupled to various types of equipment for individual separation and/or determination (e.g. hydride generation-atomic fluorescence detection [44], electrochemical detection [279] and high performance liquid chromatography-hydride generation-inductively coupled plasma mass spectrometry [278]) following leaching.

5.5.3. Microwave-assisted sample drying Drying is defined as the removal of volatiles from a sample. One common problem associated with the process of assaying in analytical laboratories is obtaining precise, reproducible methods of sample drying. This step should result in identical sample compositions irrespective of differences in starting volatile content and sample history. Usually, the major volatile component to be removed from a sample is water. Different sample types absorb variable amounts of water and therefore must be dried to an equilibrium weight. When a plot of weight loss versus time reaches a plateau, equilibrium weight has been achieved. Many samples are hygroscopic and must be kept in a moisturefree environment (e.g. that provided by a desiccator) after drying. Variation in water content is one of the most common sources of irreproducible sample weights. This can result in decreased assay precision and accuracy, and possibly give biased analytical data. In order to avoid or at least minimize the problem, samples are usually dried before weighing. Classical conventional methods of drying include the use of convection ovens, vacuum ovens and desiccators. All three methods are effective and can provide samples with reproducible composition. However, some samples may contain different temperature-dependent volatile components or exhibit variable degrees of hydration. This


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can lead to varying equilibrium weights and should be of concern to the analyst. Generally, the least aggressive method yielding reproducible results and stable sample composition will be the pretreatment of choice. The least aggressive method minimizes the risk of chemical alteration of the sample. Microwave-assisted drying provides a simple, expeditious alternative to conventional drying methods. Specially designed microwave ovens are now commercially available for this purpose [197,283-286]. One example is described in Section 5.3. Because solvent molecules are directly heated by the microwave radiation, they can be volatilized without heating the container that holds them. This reduces the cooling time required in a desiccator before accurate weight measurements can be made. In concert with drying samples, moisture determinations can be performed [4,7,287,288]. One important advantage of microwave-assisted drying methods is that, under reduced pressure, water in the sample can be removed at lower temperatures, so the decomposition or loss of volatile organic compounds and the formation of local hot spots are avoided to some extent [5].

5.5.4. Microwave-assisted distillation Distillation has long been used to separate volatile chemical components. The technique is still widely employed today in a variety of industries and for a range of separations. While conventional distillation methods rely on conduction for heating, microwave heating results from direct interaction of the energy with the sample, thus allowing energy transfer to the entire sample as opposed to this taking place via convection currents as with conductive heating. This advantage makes microwave heating an excellent candidate for replacing distillations and refluxing procedures limited by long heating periods. This microwave-assisted step has been used in the distillation of inorganic arsenic from soil [11] and seafood products [63,65], as well as in that of organic off-flavour compounds from animal tissues [66,67,289-292] (using a coupled solid-phase extractor such as that of Fig. 5.12B to allow the retention and preconcentration of distilled analytes). In both cases, microwave-assisted distillation afforded a significant reduction in sample preparation time relative to conventional distillation and also reduced the amount of sample to be handled.

5.5.5. Microwave-assisted protein hydrolysis Acid hydrolysis of protein samples has been the rate-determining step in amino acid analysis ever since commercial HPLC amino acid analysers became available in the late 1960s. Microwave-assisted hydrolysis is an alternative to conventional hydrolysis that has changed this situation dramatically. Thus, protein hydrolysates can now be prepared in less time than a single chromatographic run without compromising accuracy or precision. Microwave-assisted protein hydrolysis is particularly advantageous in cases where two or more hydrolyses are performed to achieve optimum recoveries of labile and refractory amino acids. For example, microwave hydrolysis runs of 20 and 40 min duration can be performed without altering the chemistry of amino acids instead of one and two days of conventional hydrolysis.

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Early studies in microwave-assisted hydrolysis of proteins showed the recoveries of amino acids after microwave hydrolysis to be quite consistent with the results provided by conventional hydrolysis [293,294]. Engelhardt et al. used on-line microwave hydrolysis in an HPLC system followed by derivatization with OPA to improve the detection sensitivity for proteins [295]. The initial work on microwave-assisted hydrolysis of proteins was conducted using commercial domestic microwave devices. Different laboratory microwave ovens featuring various methods of operation are now commercially available as well. The microwaveassisted hydrolysis of proteins or peptides is usually performed in sealed containers [296] where the sample is in direct contact with the concentrated acid used for hydrolysis. In vapour-gas microwave hydrolysis [297], the acid is evaporated and only the vapour contacts the sample, which prevents contamination of the samples by small amounts of amino acids potentially present in the acid used for analysis. Microwave-assisted hydrolysis systems equipped with temperature regulators suitable for quality control of protein products are now available. In fact, microwave hydrolysis is the sole technique in which the acid temperature and vapour pressure are measured directly, in situ. Other devices measure the temperature of a heating unit such as metal heating block or oven surrounding a hydrolysis chamber. One major concern in the hydrolysis process is the potential racemization of amino acids. Peter et al. [298] used both microwave and conventional hydrolysis to determine the amino acid composition of three synthetic peptides and found microwave-assisted hydrolysis to result in reduced racemization and in higher recovery of sensitive amino acids than hydrolysis by conventional heating with hydrochloric acid [299].

5.6. SAFETY CONSIDERATIONS ON THE USE OF MICROWAVE ENERGY Standards, limits and tolerance ranges have been established for microwave radiation exposure in most of the industrial world. The United States, the former Soviet republics, the United Kingdom, the former Czechoslovakia, Canada, Australia and the European Community, military and governmental bodies in these countries, as well as international organizations, have all established safety standards [300,301]. An underlying reason for the large number of exposure standards in existence today is the manner in which they are defined (e.g. by the electromagnetic energy frequency, duration of exposure, body mass and time or periodicity of exposure). Research into the biological effects of microwave radiation exposure has been extensively discussed in several reviews dealing with scientific, industrial and medical applications [300-302]. Overall, the effects on human tissue are thermal in nature and relate to overheating of exposed tissue. The underlying protective principle of several standards is derived from data on the amount of energy required to raise human skin and tissue temperatures to biologically significant levels. Exposure to energy such as sunlight is basically a surface phenomenon; however, microwave energy penetrates the skin into the subcutaneous tissue and therefore also raises the temperature level of tissue and blood.


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Proper usage of laboratory microwave equipment is the responsibility of laboratory personnel. It is possible to render the safety devices of many instruments and vessels ineffective by carelessness or misuse. It is the responsibility of the analyst to follow good laboratory practices and the manufacturer's instructions when assembling, using and maintaining microwave equipment. For example, by placing a microwave system inside a fume hood, where exhausted acid fumes may get circulated around the unit, the designed physical isolation of the electronics from the cavity is defeated. Accelerated corrosion of the electronics, including the safety interlock mechanisms and control circuits, can result. Chemical vapours should always be transported away from the unit, or the cavity air swept away to an exhaust hose, a fume extraction or neutralization system, or a hood. Deterioration of the waveguide, door seals or cavity walls can provide leak paths for the escape of microwave radiation and result in degradation of the equipment. The hazards associated with inappropriate use of microwave equipment cannot be entirely prevented by interlocking devices and other safeguards. However, the risk can be minimized if the analyst continually inspects the system to ensure that the equipment is maintained in safe working order. If any portion of the microwave unit such as door seal or vessel casing becomes damaged by a serious event such as an acid spill, prolonged wear or impact, the safety of the equipment should be re-assessed before it is returned to service. In addition to compliance with microwave energy leakage standards, safety interlock devices are required to prevent accidental exposure on all commercial and consumer microwave equipment. These interlocks protect against initiating or continuing the emission of microwave energy into the cavity if the microwave system door is open or misaligned. Safety devices should never be removed or comprised on any microwave equipment, but especially on laboratory systems. Other important components for safe operation such as wavelength attenuators in atmospheric-pressure systems, door seals or waveguides should be inspected and tested for microwave leakage if corrosion is observed or a vessel vents and reagents have prolonged contact with non-resistant parts of the equipment. Other considerations such as the constituent materials of closed vessels and the cautions to be exercised in handling hazardous acids such as HCIO 4 have been addressed in detail throughout this chapter.

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Chapter 6

High-pressure, high-temperature solvent extraction 6.1. INTRODUCTION Over the past few years, a technique based on the use of solvents at a high pressure and/or high temperature without reaching the critical point has gradually emerged as an efficient means for increasing automatability, shortening process times and reducing the amounts of solvent required to digest or leach solid samples. The use of this technique has grown dramatically in recent years, as shown by the more than 150 papers published in barely six years (from 1994 to 2000). Such features as the low consumption of organic solvents and the expeditiousness of determinations make this technique especially suitable for environmental analysis, the area where it has expanded to the greatest extent. Extraction with an aqueous or organic solvent at a high pressure and/or temperature can be made by using the static mode, the dynamic mode (the solvent is circulated in a continuous manner through the sample) or a combination of both. Ever since the inception of this technique, there has been much confusion regarding the name it should be given. In 1996, Richter et al., of Dionex Corporation, published a paper in Analytical Chemistry called "Accelerated solvent extraction: a new tool for the environmental field", where they reported a new extraction methodology based on the use of organic solvents at a high pressure and temperature as a means for accelerating and increasing the efficiency of leaching processes [1 ]. The previous year, however, these authors had published two studies where they used the technique to extract PCBs and PAHs [2], and organophosphorus pesticides and various herbicides [3], from soil. Also, several 1994 internal documents of Dionex Corporation already referred to the extraction of BNAs (bases, neutrals and acids) [4], and of organophosphorus pesticides, chlorinated herbicides and PCBs [5], using a technique that the firm then called "accelerated solvent extraction" (ASE). Simultaneously to the previous studies, in September 1994 a research group at the North Dakota University headed by Professor S.B. Hawthorne published a paper in Analytical Chemistry [6] about the potential of using water at a high pressure (50 bar) and temperature (250~ for extracting compounds of different polarity based on changes in the dielectric constant of water with variations in temperature; the authors referred to this technique as "subcritical water extraction". The essential differences between the method of Hawthorne's group and that under development by Dionex at the time were the use of water instead of an organic solvent as leaching agent, and the manner in which the extraction proper was conducted. While the prototype developed by Dionex which was subsequently made commercially available performed static extraction (i.e. with no circulation of the solvent across the sample), the equipment used by Hawthorne et al. included a modified supercritical fluid extractor and

234

High-pressure, high-temperature solvent extraction

the extraction was conducted in a dynamic manner with a view to comparing subcritical and supercritical water extraction the latter required the use of corrosion-resistant equipment, which was more expensive. In principle, the commercial success of the Dionex proprietary model led many authors to adopt the term "accelerated solvent extraction" (ASE), which was also patented by the firm. So much so, that US EPA itself adopted it in Method 3545 [7], endorsed in July 1994, to refer to the extraction of volatile and semi-volatile compounds from soils, clays, sediments, sludges and waste solids by use of solvents at high pressures and temperatures. The virtually exclusive use of this term in the first few years was largely the result of the sole commercially available extractor for this purpose being that manufactured by Dionex. With time, however, alternative names such as pressurized.fluid extraction (PFE), pressurized hot solvent extraction (PHSE), high-pressure solvent extraction (HPSE) and subcritical solvent extraction (SSE), among others, have gradually replaced ASE, a commercial designation that bears no relationship to the foundation of the technique. In fact, in revising Method 3545 in December 1996, the EPA substituted ASE by PFE, which was confirmed in the November 2000 update of the method. The use of different names for the technique can lead to confusion as it may leave the impression that the names refer to different techniques rather than a single one involving extraction with solvent at a high pressure and temperature. However, authors tend to use different names for the static and dynamic modes, even though the names provide no clue as to which mode was used. For consistency, we shall use pressurized hot solvent extraction (PHSE), which is the most universally accepted designation, throughout this chapter unless the specific mode involved is stated. Adopting PFE was discarded because it might have induced even greater confusion; in fact, using the word "fluid" rather than "solvent" extends the scope to supercritical fluid extraction, which is also conducted at a high pressure. One term that accurately reflects the foundation of the technique is high-pressure, high-temperature solvent extraction, which is the title of this chapter; however, the resulting acronym (HPHTSE) is too complicated and, therefore, impractical. Although static pressurized hot solvent extraction (SPHSE) is the most suitable designation, ASE is used here to refer to the static mode because it is the acronym most widely employed at present and will therefore be much more familiar to readers; in addition, the commercially available extractor that bears this name can only perform static extractions. The dynamic mode uses water as extractant in most cases, so authors tend to refer to it as "subcritical water extraction". However, because some applications use alternative solvents, we shall henceforward refer to it as dynamic pressurized hot solvent extraction (DPHSE) in order to clearly state that it constitutes the dynamic mode. This chapter discusses the principal aspects of the technique in its two modes, the devices typically employed by each and their amenability to coupling with subsequent operations of the analytical process. Also, the main analytical applications of both modes in analytical chemistry are described, and their advantages and disadvantages with respect to alternative techniques such as Soxhlet, ultrasound-assisted, microwave-assisted or supercritical fluid extraction, discussed.

High-pressure, high-temperature solvent extraction

235

6.2. VARIABLES AFFECTING THE EXTRACTION PROCESS

The influence on the extraction process of the different parameters that affect performance depends on a number of steps that govern the transport of the analytes from the matrix to the bulk fluid. Although some details remain to be worked out, a number of steps can be envisaged to control the extraction. Thus, the desorption of analytes from solid samples can be described in the light of a model consisting of three steps, namely: (1) desorption from a solid particle; (2) d([./i~sion through the solvent located inside a particle core; and (3) solubilization into the bulk of the flowing fluid (see Fig. 6.1 ). Each step depends on a number of factors including temperature, pressure, solvent type, solvent volume, solvent flow-rate, matrix composition, sample size and extraction time, the effects and potential manipulation of which should be examined on a case-by-case basis. 6.2.1. Temperature

Temperature is the single most important parameter influencing the kinetics of mass transfer in PHSE or, in other words, the most important factor contributing to increasing

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Analytical supercritical fluid extraction

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Analytical supercriticalfluid extraction

297

changing the nature of the supercritical f l u i d - unless the fluid induces a deep physical change in the matrix. The increased suitability of polar fluids is a result of their ability to interact with the matrix and efficiently compete with the analyte for its active sites. Choosing a supercritical fluid on the basis of its Hildebrand solubility parameter is pointless when different fluids are compared; rather, it seems more appropriate to rely on the dipole moments of the fluids. Thus, based on its dipole moment, CHC1F 2 should be more suitable than CO 2 and N20 for extracting polar analytes - - the opposite conclusion is reached if their solubility parameters are compared, however.

Influence of the fluid temperature Temperature is of paramount importance in the SFE technique as it affects all extraction steps (desorption, diffusion and dissolution). The relative significance of each step depends on the particular matrix and analyte. Supercritical fluid extraction must be done at a temperature at least 10~ higher than the critical temperature of the fluid. The maximum usable working temperature may be limited by thermolabile components of the sample. The solute volatility and diffusivity increase with increasing temperature; however, the density of a supercritical fluid at a given p r e s s u r e - and hence its solvent p o w e r - decrease with increasing temperature. In addition, the temperature plays a central role in the desorption kinetics, which may determine the extraction rate of adsorbed analytes. The solubility parameter for CO 2 and solutes decreases as the temperature is raised. At high temperatures, a decrease in solubility parameter for the solute can be more significant than one in that for the solvent. All these factors have led to most supercritical CO 2 extractions being done at mild temperatures (e.g. 50~ and a pressure that is raised as the polarity of the target analytes increases in order to avoid the risks inherent in thermolabile samples. However, Langenfeld et al. [27] have shown the temperature to strongly influence the extraction efficiency for polar and strongly retained compounds in environmental matrices such as sediments. The effect of temperature can be related to a reduction in the kinetic constraints associated to overcoming the energy barrier of the desorption step, or to thermal alterations of the matrix. The rate of diffusion of the solute through the sample matrix and within CO 2 can also be increased by raising the temperature. In addition to increased solute diffusivity, an increase in CO 2 diffusivity can facilitate absorption of the fluid in a polymer and hence diffusion of solutes across it.

Influence of the presence of a modifier The solvent characteristics of a supercritical fluid can be altered by adding a modifier (also known as an "entrainer" or "cosolvent"). The mechanism of action of the modifier depends on both the type of matrix concerned and the form in which the analytes occur in it. A modifier can have four different effects, namely: (a) increase the analyte solubility by interacting with the solute in the fluid phase; (b) facilitate solute desorption by interacting with bound solutes, the matrix active sites or both; (c) favour diffusion of the solute within the matrix; and (d) hinder diffusion of the solute within the matrix through contraction, which will result in decreased recovery.

298


Fig. 7.6. Influence of various modifiers on the extraction efficiency for PAHs. 1 fluoranthene, 2 pyrene, 3 benzo(a)anthracene, 4 chrysene, 5 benzo(b)fluoranthene, 6 benzo(k)fluoranthene, 7 benzo(a)pyrene, 8 indeno(1,2,3-cd)pyrene. (Reproduced with permission of Pergamon Press.) Modifiers very often accompany carbon dioxide as supercritical extractant. Because CO 2 is non-polar, it is classified as a non-polar solvent; however, because of its large molecular quadrupole, it exhibits some affinity for polar solutes. Although modifiers are added to supercritical fluids to increase their polarity, they can also impart decreased polarity, aromaticity, chirality and the ability to further complex organometallic compounds. Just as carbon dioxide is the most popular substance for use as a supercritical fluid, it is also that to which modifiers are most frequently added. This is so because modifiers are seen as the means for enabling the use of CO 2 in situations where it may not be the best solvent. For example, methanol is added to supercritical CO_, to increase its polarity, aliphatic hydrocarbons to decrease it, toluene to impart aromaticity, [R]-2-butanol to add chirality and tributyl phosphate to enhance the solvation of metal complexes. The amount of modifier to be added depends on the properties of the extractant and those of the analyte and matrix; usually, it ranges from a few

Analytical supercritical.[tuid extraction

299

TABLE 7.4 CRITICAL TEMPERATURE AND PRESSURE FOR SELECTED SUBSTANCES USED AS MODIFIERS OF CARBON DIOXIDE Substance

T. (K)

P~ (bar)

Methanol Ethanol

513 514 537 508 536 508 546 593 467 510 536 508 562 592 742

81 61 51 48 42 47 48 58 36 63 54 30 49 41 24

l-Propanol 2-Propanol 3-Propanol Acetone Acetonitrile Acetic acid Diethyl ether Dichloromethane Chloroform Hexane Benzene Toluene Tributyl phosphate

microlitres to 5-10% of the main extractant Cleland et al. [28], however, added as much as 20% of methanol to CO 2 to recover arsenic from dogfish muscle. Figure 7.6 illustrates the effect of an identical amount (200 ~tl) of various modifiers added to supercritical CO 2 for the extraction of PAHs. The volume of methanol added to the sample can dramatically alter the extraction efficiency [29]. Thus, the addition of methanol resulted in drastically reduced recoveries in the extraction of vitamins D 2 and D 3 from pharmaceuticals; also, toluene had no significant effect, whereas 0.25 ml of diethyl ether increased the recovery by more than 10% [30]. The influence of modifiers on SFE performance has been [31 ] and continues to be documented [32-34]. The organic solvents most frequently used as modifiers for supercritical CO 2 are shown in Table 7.4. In addition to its nature and concentration, the way the modifier is added (and the imbibition and equilibration times) must be considered in optimizing its usage. The manner in which the modifier is supplied, whether static or dynamic, is dictated by its mechanism of action, the available equipment and fluid costs. In the static addition mode, a preset volume of modifier highly dependent on sample size is directly added to the sample, held in the cell, prior to extraction. The principal disadvantage of this mode is that, as the supercritical fluid begins to circulate through the sample, the modifier is swept from the extraction cell, so the matrix is brought out of contact with the modified supercritical fluid. Although dynamic addition is usually more effective than static addition the modifier is continuously passed through the sample in the former - - Ashraf and Taylor [35,36] found the static addition of methanol

Analvtical supercritical.fluid extraction

300

to CO 2 to be more effective in the extraction of PCBs from river sediments. Dynamic addition can be done by using pre-mixed fluid reservoirs or by mixing the pure components in the propulsion system, whether by placing the liquid modifier in the cylinder of a syringe pump or by using two pumps (one for the fluid and the other for dispensing the required amount of modifier). Most SFE applications use methanol as modifier. In some, however, alternative cosolvents such as hexane, aniline, toluene and diethylamine have proved more effective.

Influence of the extractant volume The influence of the volume of extracting fluid used is exerted mainly through the solubility of the target analytes in the supercritical fluid. Such a volume must be optimized in terms of the flow-rate, which will allow one to determine the influence of both in addition to that of the analyte solubilities and extraction kinetics.

7.4.2. Properties of the analyte The solubility of a solute in a supercritical fluid is dictated by its polarity, volatility and molecular weight. Thus, the less volatile and more polar a given analyte is, the less readily soluble in a non-polar extractant such as supercritical CO 2 it will be. Analytes of a high molecular weight containing polar groups call for high fluid densities and modified supercritical CO 2. On similar polarity, the extraction efficiency decreases with increase in analyte molecular weight. Figure 7.7 illustrates the influence of the molecular weight of PAHs on their extraction at two different efficiency levels [20]. An identical trend is observed in both cases that is consistent with the solubility of PAHs in

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301

supercritical CO 2 (this decreases with increasing molecular weight of the solute). By way of example of the differential behaviour of extraction depending on both the nature of the solutes and that of the extractant, the SFE efficiency achieved with CHCIF 2 does not depend on the molecular weight of PAHs; by contrast, the efficiency with which PCBs are extracted by supercritical CO 2 and N20 usually increases with increasing molecular weight of the solute [37]. The highest possible solubility is achieved when the solubility parameter for the extracting SF is similar to that for the solute. The extraction conditions must be chosen in such a way that the solute solubility in the fluid will be maximal whenever a large amount of solute (a major constituent) is to be extracted. The influence on the extraction efficiency of the analyte concentration in the target sample increases as the analyte solubility decreases.

7.4.3. Properties of the sample The most influential characteristics of the sample in relation to SFE are the nature of the matrix and its porosity, moisture content, surface-to-volume ratio and size.

Influence of sample size Increased amounts of sample call for proportionally increased volumes of supercritical fluid. The sample volume and extraction time can have strong effects in those cases where the analyte concentration in the sample is quite high. Figure 7.8A illustrates the recovery of PCBs from river sediment achieved with pure supercritical CO 2. As can be seen, recovery of the target analytes was almost quantitative after 50 min of dynamic extraction of 100 mg of sample; on the other hand, quantitative extraction from 1 g of sample was impossible even after 120 rain, possibly because the extractant was saturated with PCBs [38].

Influence of particle size The SFE rate increases with decreasing particle size and increasing surface area-toweight ratio. Figure 7.8B illustrates the influence of the former variable on the SF extraction of cocaine from coca leaves [22]. As can be seen, quantitative extraction of cocaine from samples with particle sizes over the range 220-470 lam was impossible even after 60 min; on the other hand, samples with particle sizes of 150-170 ~tm were quantitatively extracted within 15 min.

Influence of the nature of the matrix The extraction efficiency depends strongly on the nature of the matrix; as a result, the best supercritical fluid in terms of analyte solubility may not also be the most efficient. Differences in extraction efficiency for a given analyte under identical conditions arise from the matrix. The presence of functional groups on the surface of the matrix or in its components and their ability to bind to the analytes, the organic matter and moisture

Analvtical supercritical fluid extraction

302

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Analytical supercritical )quid extraction

303

In field-treated matrices, however, some pesticide bound to lignin in the plant may remain in the matrix [39].

Influence of sample moisture The effects of this variable depend on both the matrix and the analytes. Thus, hydrophobic matrices facilitate penetration of supercritical CO2; also, because the fluid is water-immiscible, the presence of moisture can make the analytes inaccessible to it. Figure 7.9A illustrates the influence of this variable on the kinetics of extraction of pyrene from natural sludge, both as collected (45% moisture) and after air-drying (2% moisture) [40]. However, water added to the sample can facilitate extraction (e.g. that of caffeine from coffee beans, Fig. 7.9B). Natural samples with a high moisture content can plug restrictors as a result of the water they contain freezing at restrictor tips. This problem can be overcome by raising the restrictor temperature, at the expense of losses in the more volatile analytes. Removal of fat in the sample together with the analytes can hinder collection of the analytes and require clean-up of the extract prior to analysis.

7.4.4. Dynamic and geometric variables Dynamic factors are among the key variables to be optimized in an SFE process. In addition to extracting the analytes, the primary function of the supercritical fluid is to transport the solutes to the collecting vessel or to an on-line coupled chromatograph or detector. Ensuring efficient transportation of the analytes following separation from the matrix entails optimizing three mutually related variables, namely: the flow-rate of the supercritical fluid, the characteristics of the extraction cell and the extraction time. These factors must be carefully combined in order to allow the flow-cell to be vented as many times as required.

Influence of the.flow-rate In some cases, the flow-rate is the single factor most strongly influencing the extraction efficiency: the higher it is, the greater the amount of analyte that is extracted over a given interval [38]. In other cases such as the extraction of fluoranthene from soil, the flow-rate scarcely affects the results above a 0.3 ml/min level [41]. The optimum conditions for a particular extraction cannot be established on the basis of the number of times the extraction cell is vented because the extraction of most analytes is kinetically limited. Occasionally (e.g. in the extraction of cocaine from coca leaves), the process is governed primarily by the solubility of the analyte rather than by its desorption kinetics (see Fig. 7.10) [22].

Influence of the extraction cell The size, geometry and void volume of the extraction cell, in addition to its amenability to decreasing compaction by stirring or ultrasonication, all influence the SFE process.


304

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EXTRACTION TIME, min Fig. 7.10. Influence of the flow-rate on the extraction of fat from potato chips using supercritical CO2 at 340 bar at 60~ (Q) 0.7 ml/min, (11) 1.4 ml/min, (A) 1.9 ml/min, (V) 2.6 ml/min. (Reproduced with permission of the American Chemical Society.) As a rule, the volume of supercritical fluid passed through the cell in one extraction should be at least ten times higher than that of the cell; in this way, if the inner volume of the cell is reduced (e.g. by inserting a piece of an inert material), the extractant passing through it will be renewed more frequently. The effect of the cell void volume varies dramatically depending on whether the extraction is dynamic or static in nature [42]: while a high void volume favours static extraction (it allows more fluid to be held in the cell and the analytes to be more extensively solvated), a high cell volume can lead to incomplete extraction in the dynamic mode as a result of the flow velocity being inadequate to sweep the analytes when the sample deposits on the cell walls. The linear velocity of the supercritical fluid is probably the most influential factor related to cell size. Figure 7.11 illustrates the influence of this parameter on the extraction of two dioxins [43]. As can be seen, the smaller cell provided the better results in both instances. The influence of cell geometry has been found to vary widely depending on the form (natural or spiked) in which the analytes are present in the matrix. At a constant cell inner volume, the effect of cell geometry on the extraction efficiency for natural samples such as lemon or soil is negligible provided elongated cells of small inner diameters or wide, short cells are used [31]. Conversely, recovery from spiked samples decreases with decreasing inner diameter-to-length ratio [44,45]. While the orientations of the flow and cell have minimal effects on the extraction rate for PAHs, the cell dimensions strongly influence their recovery.

Influence of the extraction time As a rule, natural samples require longer times for quantitative extraction of analytes than do spiked samples. Thus, complete extraction of cholesterol from egg yolk takes


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60 min compared to only 30 min for the quantitative extraction of this substance added to glass wool [18]. Even with natural samples, the time required to extract a given analyte varies markedly depending on the type of matrix that contains it. One case in point is the extraction of PAHs from certified reference materials (CRMs) with a matrix consisting exclusively of soil [46].

7.4.5. Analyte collection modes The effectiveness of the different ways in which the depressurized mixture emerging from the restrictor can be collected depends on the characteristics of the particular extractant and analytes. When the extractant is a gas under atmospheric conditions (e.g. CO2), the three collection modes described in Section 7.3 are applicable.

Solvent bubbling The temperature of the collecting solvent is one of the most influential variables in this context as it dictates to what extent the solvent will evaporate or be swept by evolved gas bubbles. The temperature of the bubbling system is altered by effect of the restrictor end which is usually kept at a high temperature to avoid plugging through saturation being immersed in the solvent, and of the cooling resulting from expansion of the supercritical fluid. The collection efficiency can be boosted by altering several experimental variables (e.g. by using a vial with a small inner diameter to increase the distance travelled by the bubbles in a given collector volume or by placing a filter at the restrictor outlet in order to reduce bubble size).


307

Sorption This collection mode entails the use of an appropriate solid, either in the line or at the restrictor outlet, whether packed in a column or as a bed. The material used for this purpose should ensure proper retention with minimal or no analyte losses, and enable the use of the best possible type and volume of eluent to desorb the target analytes. When the extracted analytes differ in polarity, a mixture of sorbents must be used to ensure adequate collection efficiency. Comparative studies of potential sorbents for SFE have been conducted, using the same or different fluids as extractants. Thus, the extraction of sulfonamides from chicken liver using supercritical trifluoromethane or CO2, both containing methanol as modifier, was followed by off-line collection on neutral alumina or C~s and a 1"1 phosphate/methanol rinse with small differences in performance among sorbents [47]. Subsequent experiments using variably sized grains of silanized glass, synthetic silica beads, C~s-modified silica, fused silica beads, cyano-modified silica and mixtures thereof, as well as cryogenic trapping on stainless steel balls, for the collection of compounds such as acetophenone, N,Ndimethylaniline, naphthalene, 2-naphthol and n-tetracosane following SFE with modified CO 2 revealed the best collecting material to be a C~s-silica bead mixed phase [48]. Supercritical fluid modifiers alter the behaviour of the sorbent materials, and hence their performance, by changing or saturating their active sites. The effect can be checked experimentally. Thus, the extraction of dinitroglycerin and trinitroglycerin retained in granulated active coal with pure CO 2 and the same fluid containing 2% methanol, followed by collection in a C~s sorbent, was found to be much more efficient with the modified supercritical fluid" 9.9% versus 2.9% for dinitroglycerin, and 97.3% versus 89.6% for trinitroglycerin [49]. The improved efficiency, however, could not be unequivocally ascribed to the presence of the modifier in the extraction step or the collection step. Mulcahey and Taylor studied the influence of the trap temperature on the collecting efficiency for a series of organic compounds. Figure 7.12 exposes the adverse effect of the modifier (4% and 8% methanol added to supercritical CO 2) on the collection of acetophenone and N,N-dimethylaniline on ODS [50].

Cryogenic trapping This collection mode involves deposition on stainless steel by the sole effect of cooling the restrictor and pointing its outlet at metal balls. As a result, its performance is markedly temperature-dependent. Experiments with open and sealed collection systems have revealed the former to be subject to much more significant losses [51]. Obviously, the volatility of the target analytes plays a crucial role also in this step and, as such, must be carefully optimized.

7.5. APPROACHES TO IMPROVING SFE PERFORMANCE

Although supercritical fluids are widely regarded as "supersolvents", this designation is utterly unrealistic, as one can easily see by comparing their solvent powers with


308

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Analytical supeJvritical fluid extraction

309

TABLE 7.5 APPROACHES TO IMPROVING SFE PERFORMANCE Altering the properties of the supercritical fluid Using a polarity modifier Using an alternative supercritical fluid Ion-pairing

9 9 9 9

Esterification and related reactions Formation of organometals Chelation Micellization

those of conventional liquids. In any case, such features as increased solute diffusivity, decreased viscosity and increased solvent strength, all of which are easier to control than in liquid solvents, have promoted the use of supercritical fluids for treating solid samples. Most analytical supercritical fluid extractions have focused on CO2 as fluid on account of its high inertness and purity, low toxicity and moderate cost. Although CO 2 is an excellent solvent for non-polar organics, its polarity is occasionally too low to ensure efficient extraction, either because the analytes are poorly soluble or because the extractant is unable to displace them from the active matrix sites. In any case, the potential of CO 2 for analytical SFE has been exaggerated, mainly because of its excellent performance in the extraction of analytes spiked to inert supports. The far from natural behaviour of these solid samples has led some to consider supercritical CO 2 the ideal SFE leacher. However, matrix-analyte interactions dramatically reduce its extraction efficiency, the effect increasing with increasing polarity of the analytes. The performance of SFE has been improved since its inception by circumventing the shortcomings that hinder quantitative leaching of polar and ionic species. The ensuing modifications have ranged from the mere alteration of extractant characteristics such as pressure and temperature to the use of complex derivatization sequences and include the manipulations shown in Table 7.5, which are used to raise the polarity of the extractant and lower that of the target species. The effects of changing the physical properties of the supercritical fluid and using a modifier are discussed in Section 7.4, so only the other approaches are dealt with here. 7.5.1. Using an alternative supercritical fluid One way of improving SFE efficiency is by using a more suitable SF to extract the target analyte. Unfortunately, the choice of fluids other than CO 2 is restricted by the desire to have reasonable critical parameter values and costs, chemical inertness, low toxicity and little environmental impact. The use of supercritical N20 has proved to increase the extraction efficiency for high-molecular weight PAHs and chlorinated dibenzo-p-dioxins from fly ash and sediment [52]. This extractant, however, does not always improve the extraction efficiency [53]; also, it can be explosive in the presence of reactive organics. Other polar fluids such as CHCIF 2 (Freon-22) have exhibited increased efficiency in the extraction of nitrated and non-nitrated PAHs from particulate matter in diesel exhaust [54]. Freon-22 has also been found to allow significantly fast and effective extraction of

310

Anah,tical supercritical fluid extraction

steroids relative to CO 2 [55]. A comparison of Freon-22, N20 and CO 2 as supercritical fluids for the extraction of native pollutants including PCBs from railroad bed soil showed the first to be the most efficient most likely as a result of its high dipole moment. Trifluoromethane has also been tested as an extraction solvent for sulphonylurea herbicides [56]. Unfortunately, the high environmental impact of these alternative extractants significantly detracts from their use. Supercritical methanol is one other excellent solvent but is less attractive to use because it is liquid under ambient conditions, which complicates sample concentration following extraction [57]. Supercritical ammonia may also be an attractive solvent as regards strength [58] but is difficult to propel because it tends to dissolve pump seals, is chemically reactive and probably too hazardous for routine use. One of the most promising alternatives to CO 2 is supercritical water. Like CO,,, water is an environmentally acceptable solvent, but has not yet received attention as an analytical extractant for environmental solids because, in contrast to CO 2, water is too polar to efficiently leach most non-ionic organics associated to environmental solids. As can be seen in Table 7.2, water has a dielectric constant of ca. 80 at ambient temperature and pressure; fortunately, however, the constant can be substantially lowered to accommodate much milder and potentially useful conditions simply by raising the temperature at a moderate pressure (see Fig. 7.13, which shows that water can be made even less polar than CC14). Thus, simply by changing its temperature, the solvent polarity of water can be expanded over a very broad range. By contrast, the dielectric constant of supercritical CO, ranges from 1 to 1.6 only. As shown by Hawthorne et al. [59], water can be

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336

Analytical supercritical lquid extraction

50-79% for another 33, between 20% and 49% for a further 8, and less than 19% for the remaining 2. All three techniques provided unacceptable results for 15 polar compounds, 10 of which, however, were recovered by more than 70% using MAE with acetonitrile at 115~ Again, these results lead to the conclusion, largely demonstrated when more drastic extraction techniques are compared, that the energy used by SOX (viz. that of the condensed solvent exclusively) is inadequate to break strong analyte-matrix bonds; this makes SOX unsuitable as a model technique. One special application of SFE involves its joint use with subcritical water to extract Dacthal and its acid metabolites from soil; the herbicide is converted to monoacid and diacid derivatives that are more readily soluble in water than the parent compound. Thus, following extraction of Dacthal with supercritical CO, at 400 bar at 150~ for 15 rain, its monoacid and diacid metabolites were extracted from soil in 10 min, using subcritical water conditions (viz. 200 bar and 50~ The metabolites were trapped in situ on a strong anion-exchange disc placed over the exit frit in the extraction cell and subsequently combined with Dacthal by placing the disc into the GC autosampler vial containing the SFE extract to allow their simultaneous elution from the disc and derivatization to their ethyl esters. Only a single sample was analysed; also, because the disc-catalysed alkylation reaction did not transesterify Dacthal, its speciation was maintained [180].

Comparison of PAH extraction methods The extraction of PAHs is worth separate discussion here on account of the vast number of applications it has e l i c i t e d - over 130 literature references, most involving soil samples relative to other species. A comparison of SFE and SOX for the removal of 11 PAHs from soil using neat CO 2 revealed the latter technique to require eight hours to match the efficiency achieved in one with the former [181]. One application involving the analysis of polluted land samples using both neat and modified supercritical CO 2 revealed the presence of a modifier and the extraction time to be the two operating variables most strongly influencing the recoveries of 16 PAHs. A subsequent comparison of the use of SFE for 1 h, MAE for 20 rain and SOX for 6 h showed them to provide extraction efficiencies of 458.0 (RSD 3.1%), 422.9 (RSD 2.4%) and 297.4 mg/kg (RSD 10.0%), respectively. The precision of SFE was found to be decreased by the presence of sulphur in the soil [182]. In one other comparison of SFE, SOX and MAE involving the extraction of 17 PAHs from a native polluted soil, the first was also found to provide better results (viz. a PAH content of 458.0 mg/kg in the extract after 5 min of static extraction plus 1 h of dynamic extraction with supercritical CO 2 containing 20% methanol) than MAE with 100 ml of dichloromethane for 20 min (297.4 mg/kg) and SOX with acetone or dichloromethane for 6 h (422.9 mg/kg). The shortest time needed with MAE was partly offset by the time the vessel had to be left unopened until the control vessel reached atmospheric pressure (about 30 min) [183]. The effect of the presence of a modifier, which is critical in the supercritical fluid extraction of PAHs, has been thoroughly studied and compared with that on Soxhlet extraction. The modifier effectively displaces the analytes from the active sites in the matrix, thereby dramatically shortening the extraction time. A series of binary modifiers


337

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Fig. 7.20. Extraction efficiency achieved in the SFE of PAHs from fly ash. (A) By adding various binary modifiers to CO~ [bars represent the following combinations from left to right: trifluoroacetic acid (TFA) in toluene, triethylamine (TEA) in toluene, isopropylamine in toluene, pure toluene, tetrabutylammonium hydroxide in methanolt-toluene, citric acid in a methanol-toluene mixture and the absence of modifier]. (B) By successive application of a liquid modifier (four times) compared to Soxhlet extraction (shaded, left bars) and conventional SFE with COJpure toluene (right bars); COJTEA in toluene (-O-) and COJTFA in toluene (-A-). Fluo fluorene, phen phenanthrene, Anth anthracene, F fluoranthene, Py pyrene, B(a)A benz(a)anthracene, Chry chrysene, B(b)F benzo(b)fluoranthene, B(k)F benzo(k)fluoranthene, B(a)P benzo(a)pyrene, D(ah)A dibenz(a,h)anthracene, B(ghi)P benzo(g,h,i)-perylene, I(cd)P indeno(1,2,3-c,d)pyrene. (Reproduced with permission of Springer-Verlag.)

338


were tested for the extraction of fluoranthene (F), pyrene (Py), benz(a)anthracene [B(a)A], chrysene (Chry) and benzo(b)fluoranthene [B(b)F]; the results are shown in Fig. 7.20A. Binary modifiers were found to provide better results. A comparison with the Soxhlet extraction of a variety of PAHs in fly ash, sediments and sewage sludge was made. As can be seen from Fig. 7.20B, SFE required successive modifier applications to the extraction chamber, each followed by 10 min equilibration at 90~ and 30 min SFE with neat CO,, which made the process rather labour-intensive and time-consuming [184]. The use of methanol as modifier and BF 3 as derivatizing reagent was found to increase the extraction efficiency [185]. The extraction of PAHs with a supercritical fluid similar to that used for the Soxhlet extraction of these compounds (viz. CO 2 or dimethyl ether, either pure or modified with toluene) revealed toluene-modified CO 2 to provide an overall extraction yield of 43 mg/kg compared to 50 mg/kg with toluene-modified dimethyl ether and 32 mg/kg with SOX [186]. The ASE and SFE techniques were compared mutually and with SOX, USE and methanolic saponification extraction (MSE) in terms of efficiency in the removal of PAHs from marine sediments. The best results, which were provided by SFE, MSE and SOX, are shown in Figs 7.21A, 7.21B and 7.21C, respectively; such results correspond to different types of sediment, which again confirms the strong influence of the nature of the matrix on the extraction efficiency [187] and is consistent with more recent findings of Benner [46]. All PAH extracts from complex matrices required clean-up prior to the isolation and separation of their components. Solid-phase extraction (SPE) is nearly always required prior to the supercritical fluid extraction of PAHs from liquid samples; alternatively, samples can be spiked to a solid material such as diatomaceous earth. A comparison of SPE, SFE, SPE-SFE and liquid-liquid extraction (LLE) for the removal of PAHs from drinking water revealed the SPE-SFE combination to provide the best results and LLE the poorest [188]. Recently, the supercritical fluid extraction of PAHs was used to correlate the contents thus obtained with the bioremediation behaviour of the target analytes in a field treatment plot for 1 year; based on the results, mild SFE was deemed an expeditious, useful tool for predicting the bioavailability of PAHs in polluted soil [189].

Use of supercritical water as extractant The special features of the uses of supercritical water justify dealing with them in a separate section. No commercially available or laboratory-made extractor can operate in a continuous manner under the conditions required by supercritical water. In fact, most of the few reported applications allegedly involving supercritical water extraction actually used subcritical water conditions with not more than two extractions in the supercritical state; this can readily be inferred from the equipment used I stainless steel SF extractors can hardly withstand the drastic conditions needed for work with supercritical water. As a

Fig. 7.21. Comparison of various techniques for the extraction of PAHs, using linear regression and the following CRMs: HS-6 (A), SPM I (B) and SPM II (C). (F-l) SFE, (O) MSE, (+) ASE, (x) SOX and (A) USE in the three figures [by exception, crosses correspond to SOX in C]. (Reproduced with permission of the American Chemical Society.)


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Analvtical supercritical.fluid extraction

result, the performance of supercritical H20 has not been examined as thoroughly as that of supercritical CO~. The Research Centre of Karlsruhe conducted tests with a view to finding materials capable of withstanding the oxidizing, corrosive conditions involved in the use of supercritical water. The materials examined included various alloys such as austenite, AISI type 316 SS austenitic CrNi stainless steel and several Ni-based alloys (viz. Inconel 625, Hastelloy C-276, Nicrofer 5923, Nicrofer 6025 and Haynes alloy 214) that were used to fabricate pressure tubes for exposure to supercritical water containing 0.5-5 mol OJkg and/or 0.05-0.5 mol HCl/kg over a period of 150 h (a preliminary step on this line) [ 190]. Most research work on the use of supercritical water has been conducted batchwise and involved non-analytical determinative applications. Thus, supercritical water oxidation (SCWO) was proposed as an alternative treatment for hazardous waste disposal [191] and also as a commercial tool for decomposing trichloroethylene, dimethyl sulphoxide and isopropyl alcohol on a pilot plant scale [192]. Current commercially available equipment (the aqua Critox" system) is usable with industrial and municipal sludge, mixed (radioactive and organic, liquid and solid) waste and military waste. This commercially available treatment has a number of advantages, namely" (a) because it uses an on-site treatment method, it avoids the need to transport hazardous materials; (b) it ensures complete destruction of organic wastes and allows reuse of the effluent as process water with results that meet the regulations for drinking water; and (c) no licence for effluent or air emissions is needed. Organic reactions in supercritical water are very fast. The dehydrogenation of lactic acid to acrylic acid in a Hastelloy C-276 annular reactor at 310 bar, 320--400~ and residence times of 25-110 s in the presence of various catalysts was used to examine three main pathways [193]. Alkylation of isobutane with light olefins for the production of gasoline with a high octane number [194], the hydrolysis of vegetable oils [195] and organic synthesis [196] are among the reported uses of supercritical water. However, supercritical water does not seem to be an effective extractant as, only in special situations, are the analytes so strongly retained by the matrix to require supercritical water conditions - - under which the sample is highly likely to be degraded anyway.

7.8. PRESENT AND FUTURE OF SUPERCRITICAL FLUID EXTRACTION After the initial euphoria of the late 1980s and 1990s, SFE has consolidated as a powerful tool for the analysis of environmental, pharmaceutical, polymer and, especially, food samples (particularly fat analyses, which have boosted SF extractor sales recently) [17]. Notwithstanding its major restrictions (especially in relation to the extraction of polar analytes and the treatment of natural samples), SFE can undoubtedly expedite the pretreatment of solid samples with the aid of special approaches to the extraction of medium-polar and polar analytes. Foreseeing the direction in which SFE applications will move in the future entails considering the nature of its latest uses. Coupled supercritical CO2-subcritical water systems may facilitate the class-selective extraction of non-polar substances and families of compounds of different polarity by adjusting the temperature of the water.

Analytical supercritical.[tuid extraction

341

The development of new analyte collection methods using decreased amounts of solvent and hence providing more concentrated e x t r a c t s - is one other focus of current SFE research [43]. One other potential trend for SFE aims at field work. So far, only a single SFE method for the field extraction of PCBs and PAHs from soil appears to have been reported; the method is used in combination with GC-ECD or FID and requires no clean-up following extraction [197]. A portable system developed by the US Department of Defense is currently at the testing stage. The ensuing method uses dry ice ~ thus avoiding the need for compressed CO 2 tanks and has proved effective in the extraction of non-polar analytes; however, it requires the presence of modifiers to extract polar analytes. Such techniques as ASE, MAE and microwave-assisted SOX have lately experienced major breakthroughs [198-200]; consequently, SFE should only be considered the technique of choice for extraction purposes in those cases where its advantages over the previous alternatives (viz. cleanliness, expeditiousness and simplicity) are bound to offset the potential shortcomings arising from its inability to efficiently process specific matrices or analytes.

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Chapter 8

Devices for solid sample treatment prior to introduction into atomic spectrometers: electrothermal devices and glow-discharge sources

8.1. INTRODUCTION Atomic techniques have proved to be highly powerful tools for determining metals in a wide variety of matrices, particularly since the inception of special devices that have substantially improved on the atomization efficiency of flames. The use of electrothermal [1] and non-electrothermal devices to perform the steps involved in converting the target elements into the required atomic states has endowed absorption, emission and fluorescence atomic techniques with their present roles in analytical chemistry. Although mass spectrometry is not an atomic technique, it is also widely used for the determination of metals. For this reason, it is also dealt with in this chapter alongside the atomic techniques typically employed following atomization of a sample. This chapter is thus concerned with the sampling devices used prior to introduction into absorption, fluorescence emission and mass spectrometers. The former two always require that the target analyte be in the atomic state before the absorption or excitation source comes into play and hence the use of an electrothermal atomizer (ETA); the latter two only require vaporization of the sample (by means of an electrothermal vaporizer, ETV) if the atomic or ionized state can be reached by using a flame or plasma, vaporization-atomizationexcitation (using a glow-discharge source) in emission spectroscopy and vaporizationatomization-ionization in mass spectrometry. None of these detection techniques is dealt with as such here, although they appear in some schemes illustrating their role in specific applications. As in previous chapters, readers are referred to monographs on these wellestablished techniques for further details [2-11]. In addition to its well-known advantages over sample dissolution (viz. reduced sample pretreatment and thus increased expeditiousness in the overall analytical procedure; low contamination risks, which is an essential requirement in determining trace metals; decreased analyte losses through sample pretreatment or retention by insoluble residues; and avoidance of corrosive or hazardous chemicals), solid sampling overcomes some of the shortcomings arising from the way liquid samples are introduced into flame or plasma spectrometers. Thus, liquid sampling requires relatively large volumes of solutions (at least 5 ml for analysis under typical operating conditions). This frequently calls for dilution of the sample. Pneumatic nebulizers have the additional drawback that usually less than 5% of the solution actually reaches the flame or plasma. Ultrasonic nebulizers are more efficient but are expensive and highly sensitive towards high acid or salt concentrations.

348

Devices fi~r treatment prior to introduction into atomic spectrometers

Also, the insertion of solvents particularly into plasmas J causes a number of spectral interferences and, at dissolved solid concentrations above ca. 1%, signal suppression and salt build-up on the nebulizer, the interface or the injection tube of the torch. Solid sampling devices have been used to either atomize samples into absorption and fluorescence spectrometers or to vaporize them into flames and plasmas prior to emission or mass detection. The most usual types of devices used in this context are as follows: (a) electrothermal vaporizers (ETV) and atomizers (ETA); (b) glow-discharge (GD) sources; (c) laser ablation devices; (d) laser-induced breakdown devices; (e) direct sample insertion (DSI) devices; and (f) arc nebulizers. Because these devices have received dissimilar attention by users, they are discussed here to an extent proportional to the interest they have aroused. Thus, electrothermal atomizers and glow-discharge devices are the main subjects of this chapter, which also provides a brief description of direct sample insertion devices and arc nebulizers. Such a rapidly growing field as laser sampling warrants separate coverage and is thus the subject matter of Chapter 9. Atomizers and glow-discharge devices feature a key difference: the sample is introduced in the former and is part of the sampling device in the latter. In essence, glow-discharge sampling is more closely related to the techniques described in Chapter 9 than to electrothermal devices; however, it is discussed here both to facilitate comparisons and to balance the contents of the two chapters.

8.2. ELECTROTHERMAL ATOMIZERS AND VAPORIZERS

This section is devoted to the types of devices most frequently used for both liquid and solid sampling prior to introduction into atomic spectrometers [12-14]. Atomic techniques and mass spectrometry make massive use of electrothermal devices, the maturity of which has been endorsed by IUPAC, which has included it in its "Nomenclature, Symbols, Units and their Usage in Spectrochemical Analysis. XII. Terms Related to Electrothermal Atomization", published in 1992 and subsequently reprinted in Spectrochimica Acta [ 1].

8.2.1. Fundamentals of electrothermal vaporizers and atomizers Carbon atomizers

Some of the physical and chemical constraints on the flame atomization process which usually precluded application to solid samples were overcome with the advent of"flameless" atomization, initially accomplished with the pyrolytic coated graphite tube (or carbon rod-type) furnace atomizer. The graphite tube is a confined furnace chamber where pulsed vaporization and subsequent atomization of the sample is achieved by raising the temperature with a programmed sequence of electrical power. A dense population of ground state atoms is produced as a result for an extended interval in relation to the low atom density and short residence time of the flame. The earliest use of"furnace" devices in analytical atomic spectroscopy is credited to a simultaneous development by L'vov [15] and Massmann [16]; however, the first application of one such device to a

Devices Jbr treatment prior to introduction into atomic spectrometers

349

solid sample was reported by L'vov in 1959 [17] and followed 12 years later by the first instance of solid sampling with a commercial electrothermal atomizer [18] - - o n e year after the first commercial graphite-furnace atomizers (ETAs) were marketed by firms such as Varian and Perkin-Elmer for use in atomic emission spectrometers. Development of ETA-based solid sampling applications started in the late 1970s and early 1980s. Basically, a graphite-furnace atomizer (Fig. 8.1A) consists of a graphite tube, electrical contacts, an enclosed water-cooled housing and inert purge gas controls. The graphite tube is the usual heating element of the graphite furnace. The cylindrical tube is aligned horizontally with the spectrometer's path in atomic absorption and fluorescence techniques, and serves as the spectrometer sampling cell in AAS and AFS. The sample is placed at the centre of the tube wall onto the inner tube w a l l - which can be cup-shaped or on a graphite platform. The tube is held in place between two graphite contact cylinders that provide electrical connection. An electrical potential applied to the contact causes current to flow through the tube, the effect being heating of the tube and the sample. Quartz windows at each end of the housing allow light to pass through the tube. The heated graphite is protected from oxidation by atmospheric air by the end windows and two argon streams. An external gas flow surrounds the outside of the tube and an independently controlled internal gas flow purges its inside. The system should regulate the internal gas flow so that the internal flow is reduced or, preferably, completely stopped during atomization. This helps maximize the sample residence time in the tube and boosts measured signals. The graphite tube is heated by passing electrical current from the graphite contacts at the ends of the tube throughout its length. This type of furnace, which is longitudinally heated, is similar to the original design of Massmann [16] and is the basis for most currently available commercial graphite furnaces. It has a major liability: as the electrical contacts at each end of the tube must be cooled, there must always be a temperature gradient along the length of the tube, which results in the tube end adjacent to the electrical contacts being cooler than the central portion. This temperature gradient can cause vaporized atoms and molecules to condense as they diffuse to the cooler tube ends. This may in turn result in interferences, the most common type being incomplete removal of the analyte or matrix from the tube. Incomplete removal of the matrix during pyrolysis can increase background absorption during atomization; incomplete removal of the analyte is more serious as it results in carry-over or memory effects, whereby part of the analyte in the sample being processed remains in the tube and contributes to the analytical signal for the next. In order to reduce carry-over, most longitudinally heated furnace heating programmes use one or more clean-out steps after atomization or both increased pyrolysis temperatures and an in situ digestion procedure [19], which often entails altering the inside surface of the graphite tube (e.g. by coating with Ir sputtered in a low-pressure Ar discharge). A clean-out step involves the application for several seconds of full internal gas flow and a temperature equal to or greater than that used for atomization to remove residual sample components. While this additional step works well with the more readily atomized analytes, it does not always succeed with those that require higher atomization temperatures. The use of a high-temperatu;e clean-out step may also reduce tube lifetime. The transversely heated furnace (THGA, Fig. 8.1B) avoids many of the problems associated with the longitudinally heated furnace. The graphite tube of a transversely heated

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B)

LIGHT

1

Fig. 8.1. Typical graphite furnaces: (A) longitudinally heated model, (B) transversely heated model. For details, see text. (Reproduced with permission of Perkin-Elmer.)

Devices Jbr treatment prior to introduction into atomic .spectrometers

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furnace includes integral tabs that protrude from each side. The tabs are inserted into the electrical contacts and, when the power is applied, the tube is heated across its circumference (i.e. transversely). In this way, the tube is heated evenly over its entire length, thus eliminating or significantly reducing the sample condensation problems typical of longitudinally heated furnaces. One additional advantage of the transversely heated furnace is that it enables longitudinal Zeeman-effect background correction without the need to use a polarizer in the optical system. The comparison of a transversely heated graphite atomizer having longitudinal Zeeman-effect background correction with a Massmann-type furnace having continuumsource background correction in the determination of aluminium in nickel-based alloys by ETA-AAS revealed the THGA to be less sensitive owing to its shorter path length but also to provide better limits of detection by a factor of 2 as a result of its background correction system [20]. The Frech two-step furnace, with separate control of the vaporization and atomization functions, represents a substantial improvement on commercial Massmann-type and THGA furnaces for interference-flee analyses by ETA-AAS. However, it has the disadvantage that it relies on diffusion and convection to transport sample vapours from the cup vaporizer to the tube atomizer. Transport by purging is one solution to this shortcoming. For this purpose, the Massmann-type atomizer is heated to a steady-state atomization temperature and the THGA vaporizer is pulse-heated to have the purge gas drive the analyte from the vaporizer to the atomizer [21]. Platforms.for carbon atomizers

One source of interference with carbon atomizers arises from the analyte appearing in the furnace atmosphere before the furnace has reached the actual temperature set for the atomization step in the furnace programme. The problem results from the temperature of the tube wall rising rapidly at the beginning of the atomization step. The sample, which is in direct contact with the tube wall, immediately experiences the increase in wall temperature. When the tube wall reaches the temperature at which analyte atoms are produced, atoms are released from the hot tube surface into the inert gas atmosphere, the temperature of which lags behind that of the tube walls and is cool relative to the surface. This sudden cooling inhibits atomization of the vaporized molecular species and results in non-spectral interferences. Elimination of this delay favours the formation of free atoms and maximizes the sensitivity, which remains constant irrespective of the sample matrix. One of the earliest devices (usually platforms) used to avoid the delay was the L'vov platform, which is a flat piece made of solid pyrolytic or pyrolytically coated graphite that is placed in the bottom of the graphite tube as shown in Fig. 8.2A. When the sample is located in the shallow depression of the platform, the sample experiences the temperature of the platform rather than that on the tube wall. Since the platform is, by design, in poor physical contact with the round tube surface, the platform is heated primarily by radiant rather than conductive energy. This means that the platform will heat more slowly than the graphite tube. During the rapid heating in the atomization step, when the platform does finally reach the atomization temperature for the analyte, atoms are released into a furnace environment the temperature of which is similar to that of the

352

Devices jor treatment prior to introduction into atomic spectrometers

A)

GRAPHITE FRONT VIEW

/~ -

SIDE-ON VIEW

PLATFORM

~

(

TUBE

) TOP VIEW

B) INTERNAL GAS TEMPERATURE

ATOMIZATION FROM PLATFORM

J TIME

Fig. 8.2. Use of the L'vov platform for electrothermal atomization. (A) Scheme of the device. (B) Behaviour of the analyte as compared with the absence of the device in the atomizer. (Reproduced with permission of Perkin-Elmer.)

Devices Jbr treatment prior to introduction into atomic spectrometetw

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platform (see Fig. 8.2B). Under these conditions, dissociation is favoured and recombination minimal. The increased sensitivity exhibited by the signals of Fig. 8.2B arises from the fact that, when the furnace tube reaches a temperature at which the analyte will vaporize, the metal is driven from the surface into the gas phase. Both the temperature at which the analyte first starts to volatilize and the rate at which it does will depend on the specific nature of the matrix constituents and their amounts. Since the platform is heated by radiation from the tube walls, heating of the sample on the platform is delayed relative to the tube walls and hence to the vapour within the tube. Instead of volatilizing the analyte as the temperature changes, appropriate conditions can be found to volatilize it after the tube walls and gas phase have reached a more stable or steady-state condition. The effect of the platform is thus to delay the volatilization of the analyte until the atomizer has reached a stable (high) temperature. A number of users have attempted to improve the features of commercially available platforms as regards both shape and material with little or no success [22]. A series of additional alterations of the graphite tube atomizer intended to improve performance (e.g. the cup and cup cuvette atomizer, boat and microboat platforms, carbon rods, graphite probes, ring chambers and surface atomizers) have also been reported [23]. Interested readers are referred to specific sources [2-7] for more details. Coatings Jbr carbon atomizers"

Although graphite - - particularly pyrolytic g r a p h i t e - remains a unique material with many desirable properties for an atomizer, a number of metal coatings on the graphite substrate can be used to increase the sensitivity for some elements. Almost thirty years ago, the use of high-melting Ta and Nb carbides was proposed to raise the sensitivity of ETA-AAS for boron by using the L'vov cuvette as atomizer. Since then, interest in this idea has not declined, as shown by the more than four hundred papers on research into carbide-modified graphite tubes. Based on the structure of the carbide layer used for this purpose, modified graphite tubes can be classified into the following three categories: (a) PVD-modified tubes, where the solid layer of carbide on the surface prevents the interaction of analytes and matrices with the graphite. As a rule, these tubes have very long lifetimes and their surface activity is lower than that of the original tubes. (b) Tubes treated under atmospheric pressure with solutions of modifying elements, which distribute over the graphite surface as "islands" of the corresponding carbides. In this way, the analysed sample is kept in contact with the graphite. (c) Tubes modified under a low or high pressure with solutions or pure liquids containing the modifying elements. These tubes are placed between the previous two. After several treatments under optimal conditions, a solid carbide layer eventually forms. The tubes can also be classified as follows according to the properties of the specific carbides used in the coatings: (a) Tubes modified with metal-like carbides such as TiC, ZrC, HfC, VC, MoC and WC, which are more chemically active than pyrolytic graphite;

354

Devices fi)r treatment prior to introduction into atomic spectrometers

(b) Tubes modified with metal-like carbides such as TaC and NbC, which are more stable to oxidation by acids and possess roughly the same reactivity to oxygen as pyrolytic graphite; (c) Tubes modified with covalent B4C or SiC, which exhibit a high chemical inertia; (d) Tubes modified with compounds of the elements in the third group (La, Th and Y), the surface activity of which is scarcely dependent on the particular modifying method used. An excellent review of the methods used to modify graphite tubes; investigations of the modified surface; the physical, chemical and catalytic properties of the modified tubes; and the main processes occurring in them was published in 1998 by Volynsky [24]. N o n - c a r b o n atomizers

Although graphite furnaces are the most widely used in atomizers, alternative materials have also been employed for this purpose. Tungsten-coil atomizers have to some extent been used in connection with atomic techniques in general and ETA-AAS in particular, and their popularity continues to rise. Their high simplicity and low cost make them attractive alternatives to graphite furnaces for many applications. However, they are much more interference-prone than their graphite counterparts. The interferences experienced by W-coil atomizers have been overcome in various ways, however. Thus, Barbosa et al. [25] avoid the interference from a salt matrix and implement a preconcentration step by electrochemically reducing Pb onto the coil surface. They use a flow injection system to deliver the sample through an anode inserted in the tip of the autosampler and the W-coil itself as cathode. The use of a Wcoil as a platform inside a Massmann-type furnace provides no appreciable improvement over wall atomization [26]. Metal tube atomizers, though not available commercially, are occasionally preferred to graphite furnaces, especially for the determination of less volatile "carbide-forming" metals, mainly in liquid samples but also in solids [27]. The most salient features of laboratory-built atomizers for direct solid sampling are as follows: (a) They are usually larger than commercial atomizers, which facilitates sample insertion, prevents blocking of the radiation beam and avoids the high background signals typically yielded by solid matrices. (b) The analyte is volatilized and atomized separately. (c) They use large amounts of sample to avoid errors arising from inhomogeneous distribution of the analyte. (d) They use a low heating rate during atomization in order to avoid a high background. Atomizers for solid sampling can be heated inductively or resistively. Although resistively heated furnaces are more common, some induction furnaces can readily accommodate solid samples.

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InterJaces

The relationship established between electrothermal atomizers and detectors depends on the nature of the detector to be used for identification and/or quantitation. Thus, an absorption spectrometer uses the atomizer as its detection cell: the beam from the atomic radiation source passes through the atomizer and is absorbed to an extent dependent on the analyte concentration. Fluorescence detectors require no special adjustments for excitation of the analyte in the atomizer other than those derived from the need of collecting the emission at a right angle to the excitation source. Up to four different commercial furnaces and one tungsten furnace were used for the comparative determination of titanium by laser-excited (LE) AFS. The limit of detection for the analyte was found to be similar with all types of furnace: around 1 pg for furnaces not exposed to substantial amounts of Ti and a few picograms for those exposed to more than a few hundred picograms of this element. A commercial transversely heated graphite atomizer was found to provide the most reproducible atomization of Ti. While the W furnace exhibited no memory effects for the analyte, it gave signals that were about 250 times weaker than those of the graphite furnaces owing to incomplete atomization [28]. A special "envelope" of a commercial atomizer was designed in the form of a laboratory-built cylindrical vacuum chamber of stainless steel intended to lower the working pressure inside the graphite furnace to 7 Pa with a view to reducing matrix interferences; however, the sensitivity was dramatically affected by the high diffusion rates observed under these conditions [29]. The single 90 ~ off-axis ellipsoidal mirror fragment used as dispersive detection system in order to enhance the collection efficiency of atomic fluorescence from ETAs may be viewed as an interface between these devices and the detector [30]. The needs arising in coupling an electrothermal vaporizer (ETV) to an ICE ICP-MS or MS instrument are rather different. In the three cases, the vaporized or atomized sample must be efficiently transported to the plasma source, for excitation, or directly to the detector. In all instances, the performance of the ensuing methods is strongly dependent on the transport efficiency of the volatilized analytes from the ETV to the ICP torch or MS instrument. In other words, the vaporizer must be converted into an efficient flowthrough device. Figure 8.3A shows a commercial graphite furnace modified for minimizing transport losses of eight selected analytes; its transport efficiency was determined by monitoring a radiotracer. Figure 8.3B depicts selected interfaces used for this purpose [31 ].

8.2.2. Solid sampling modes in electrothermal vaporizers and atomizers Solid samples can be introduced into electrothermal atomizers either as such or as slurries. Each insertion mode has its own features, advantages and disadvantages. Direct sampling of the solid

With Massmann-type electrothermal atomizers, the solid sample can be introduced into the furnace either through a central hole in it or from the ends of the graphite tube. The use of this type of furnace in this sampling mode poses problems such as cumbersome

356

Devices Jor treatment prior to introduction into atomic' spectrometers

Devices Jor treatment prior to introduction into atomic spectrometers

357

sample insertion, strong spectral interferences and irreproducible deposition of the sample on the atomizer walls. Accurate positioning of the sample in these devices is important in order to avoid irreproducible vaporization resulting from the temperature gradient along the tube. Samples can be inserted in four different ways in the direct sampling mode. One involves introducing a support on which the sample is weighed; once the sample is inside the atomizer, the support is re-weighed. This procedure is used mostly with tube-type atomizers. The weighing and transfer operations require special care in order to minimize contamination and sample losses. The tantalum solid spoon commercially available from Perkin-Elmer is one device of this type. The sample can be inserted through the dosing hole of the graphite tube provided the hole is previously enlarged. Solid samples consisting of ion-exchange resins for the preconcentration of metals from solutions can be introduced into the tube by using a set of plastic tweezers and a small funnel [32]. A more advantageous approach uses a graphite support (a platform, boat, microboat, cup or probe) for both weighing and atomization. The use of inert supports made of ashless paper and cellulose acetate has also been tested [33]. However, inert supports tend to give high blank values; also, the residue remaining after atomization can degrade accuracy and reduce the atomizer lifetime. One other procedure involves introducing the sample with a special injector. In order to facilitate insertion through the sampling hole in the graphite tube, solid sample injectors operated similarly to conventional autosamplers have been used that work like a microsyringe and introduce amounts of sample ranging from 0.5-5 mg with reproducibility comparable to that of manual solid sample insertion [34]. The fourth procedure for sample introduction in the direct sampling mode uses special devices to collect the sample (by filtration through porous graphite or by electrostatic accumulation). Such devices are especially useful for solid materials suspended in gases. Samples are collected on filters made of various materials including cellulose acetate and nitrate, and PVC. Slurry sampling

Slurries for insertion into electrothermal vaporizers and atomizers are prepared by adding a liquid diluent to the solid material, previously ground, sieved when necess a r y - weighed and placed in a container ensuring stability of the slurry during the time required for sampling. The amount of solid material to be weighed depends on the concentration of the analyte and the dilution in the final volume of slurry. For less homogeneous samples, the precision is improved when the slurry is made from increased amounts of sample. The slurry sub-sample is inserted into the atomizer either by hand, using an Fig. 8.3. (A) Scheme ofa graphite-ETV modified for ICP-AES work: (1) graphite tube, (2) graphite platform, (3) electrical contacts, (4) cooling water chambers, (5) protecting gas chambers, (6) automatic shutter, (7) ETV-ICP connecting interface. (B) Two different ETV-ICP connecting interfaces: (b.1) Nozzle-type aluminium-made interface with cooling bypass gas admittance; (b.2) same as b.1 but with a larger inner diameter prior to the bypass gas entrance. (Reproduced with permission of the American Chemical Society.)

358


autosampler or with the aid of a flow injection system. When autosampler cups are used to weigh the solid material, the maximum volume of slurry is limited to the cup volume (about 2 ml), which in turn limits the maximum amount of material that can be suspended. Once the slurry is prepared, the solid sample must be evenly distributed in the volume of liquid. Slurry sampling gained increasing acceptance over the 1990s as a means for dealing with various types of samples with practical advantages over alternative procedures including the ability to use conventional liquid autosamplers, perform several replicates in a single aliquot, handle increased amounts of sample and alter the slurry concentration. Additional assets include improved analytical performance as a result of the combined benefits of solid and liquid sampling, and increased repeatability and reproducibility. On the other hand, the large number of variables to be considered complicates optimization of analytical processes involving slurry sampling. 8.2.3. Variables of solid sampling with electrothermal vaporizers and atomizers The variables that influence the solid sampling process depend on the particular sample insertion mode used. Variables affecting direct sampling of the solid Sample preparation Treatment of samples for solid sampling should be kept to a minimum in order to avoid contamination or losses - - grinding, sieving, homogenization and drying, among other operations, are often required, however. Because particle size influences accuracy and precision, samples are ground to obtain a powder or reduce the size of particles. Samples can be ground by using an agate mortar or a vibrational pulverizer. Grinding is suitable for brittle materials such as rocks, minerals, glass and soil. Also, biological materials can be converted into solids (e.g. by drying, dry ashing, plasma ashing or freeze-drying) for subsequent grinding. On the other hand, grinding metallic samples is more complicated and can introduce contamination. Particle size Most materials require grinding in order to reduce particle size to an appropriate extent (e.g. to 5 ~tm). Grinding is also recommended for solid samples that are originally in powder form when the analyte is unevenly distributed. This operation improves contact between the atomizer surface and the sample. However, it is not always easy to perform (e.g. with metallic samples) and requires great care in order to avoid contamination. The effects of grinding solid samples are somewhat controversial. In some types of sample (e.g. biological materials), particle size has no influence on accuracy but only on precision; in others (e.g. refractory materials such as rocks and minerals), vaporization of the analyte is frequently influenced by particle size. Integrated absorbance measurements for repeated atomization of the sample until thorough vaporization of the analyte have revealed that increasing the amount of sample used does not always result in a proportional increase in the atomic absorption signal. Precision is poor when the sample is inserted into the atomizer as a single particle, as found in the determination

Devices Jbr treatment prior to introduction into atomic spectrometelw

359

of Cu following preconcentration on an ion-exchange column and use of wall atomization [35].

Sample homogeneity The effect of inhomogeneity is more critical when the amount of sample is small (sample masses are usually in the range 0.1-1.0 mg). The relatively large minimum amount of sample that can be effectively handled reflects the difficulty of weighing the sample and inserting it into the atomizer. Studies of trace distributions have shown that the influence of inhomogeneity on precision is negligible with some types of sample (e.g. metals), even with amounts as small as 2 mg [36]; on the other hand, homogeneity is unimportant with samples such as rocks and glasses in amounts down to about 1 mg [37]. Sample mass The amount of sample to be introduced into the atomizer is dictated by the analyte concentration in it. The range of suitable amounts is determined first by the availability of a microbalance to handle sufficiently small masses and the ability of the atomizer to accept larger amounts. Larger graphite tubes than those commercially available have occasionally been used to introduce up to 200 mg of sample. The use of larger amounts of sample results mainly in improved precision, particularly with inhomogeneous samples. However, the range of sample masses that can be introduced into the atomizer is limited and, although amounts as small as 1 lag have been used, masses below 0.1 mg are difficult to handle (viz. to weigh and transfer). When the concentration of the target analyte is too high for the analytically useful working range of the calibration graph, one can use less sensitive lines, dilute the sample with graphite or employ an increased flow of Ar. These procedures, however, are error-prone. Vapour phase temperatures are lower when an internal gas flow is used, so matrix interferences are enhanced as a result. Analyte location in the sample The type of bond between the analyte and the solid matrix, in addition to the location of the analyte in the solid particles (viz. on the surface or occluded within) can influence absorbance measurements through differences in vaporization kinetics. One example is the determination of several metals in steel. Signals with double peaks were observed as a result of the analyte being present in two different locations of the steel grains [38]. The formation of several peaks in the atomic absorption signals was also observed in the determination of lead in plastics using a cup-in-tube device [39] and ascribed to the manner in which the plastic decomposed as the temperature was raised. Also, several peaks were observed when lead was atomized at a low pressure from copper alloys, which was also ascribed to the presence of various chemical forms of Pb or changes in its location [40]. Variables affecting slurry sampling The main variables influencing this sampling mode are slurry stability and concentration, and particle size.

Slurry stability Slurries can be maintained stable for insertion into ETAs and ETVs using various approaches, namely:

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Devices jbr treatment prior to introduction into atomic spectrometers

(a) Stabilizing agents. Slurries prepared in aqueous solutions are rarely suitable because most powdered materials undergo rapid sedimentation. This occurs immediately after the slurry is mixed and can be quantified via Stoke's law. The sedimentation rate depends on the densities of the diluent and solid material, the viscosity of the diluent medium and the radius of the sample particles. The slurry can be stabilized using a highly viscous liquid medium such as Viscalex, glycerol, a non-ionic surfactant, an organic solvent or a thixotropic thickening agent, among others. The stabilizing capability of these agents depends largely on their concentration, as well as on the characteristics and particle size of the sample. On the other hand, the maximum slurry concentration that can be used depends on the concentration of the stabilizing agent. The time interval between complete mixing of the slurry and removal of an aliquot for analysis can be extended by using a highly viscous medium similar in density to the sample particles. Problems have been noted regarding the use of viscous media to prepare slurries, however, such as the sample aliquot being inefficiently pipetted into the atomizer when a high concentration of stabilizing agent is used. Also, the sample can remain around the dosing hole, which can degrade precision with both micropipettes and autosamplers. The use of the latter additionally requires flushing the capillary between successive determinations. It is also necessary to include an additional pyrolysis step in order to remove excess stabilizing agent. Agents are usually inefficient when the slurry contains particles of a high density. Apart from stabilizing agents, the addition of wetting and antifoaming substances such as Triton X-100 can improve slurry dispersion. Stabilizing agents are the preferred choice when samples are to be inserted via an autosampler as the slurry can be allowed to stand in the sampling cups without further homogenization. (b) Magnetic stirring and vortex mixing. These were the two procedures most widely used in early work on slurry sampling. Slurries are prepared in a beaker and stirred in order to ensure homogeneous distribution of the solid material (usually for 3-5 min). The stirring action is then stopped and a sample aliquot is withdrawn for insertion into the atomizer. The sample aliquot can also be withdrawn under continuous stirring, however. The effectiveness of magnetic stirring and vortex mixing depends basically on the sedimentation rate of the suspended material and the error associated with sedimentation is a function of both particle size and the presence of particles having different compositions. As a result, large errors can be expected when the analyte is predominantly distributed in particles of a high density, which will undergo rapid sedimentation. After magnetic stirring or vortex mixing, slurries are usually introduced with a micropipette because of the difficulty of incorporating these systems into an autosampler. Some miniature stirring devices have been used in combination with an autosampler for this purpose [41 ]. (c) Ultrasonic agitation. This is by far the most effective method for homogenizing slurries for insertion into ETAs. Ultrasonic agitation can be used in combination with both manual and automated introduction of the slurry. One advantage of this method relative to magnetic stirring and vortex mixing is that the target analyte is partially extracted into the liquid phase under the ultrasonic action when slurries are prepared in an acid medium. Ultrasonic agitation is more effective than other agitation systems (see Chapter 3). Agitation with a small ultrasonic probe also allows slurries to be prepared directly in the sample cups of an autosampler, but special care is required to avoid


361

contamination when the titanium probe touches the cup wall. A device of this type is commercially available from Perkin-Elmer. The precision thus achieved depends on both the extraction yield of the analyte and the degree of homogeneity of the sample. Precision of 2-10% as RSD was obtained in the determination of Se, Pb and T1 in coal and coal fly ash, with an analysis time of less than 1 min by using ultrasonic agitation of the slurry but no pyrolysis step [42]. Commercial ultrasonic probes for slurry homogenization provide additional advantages such as increased throughput, automatability of slurry preparation and insertion into the furnace, improved reproducibility of analyte extraction, and increased representativeness of the sample aliquot. (d) Gas mixing of the slurry. Slurries can be efficiently homogenized by passing an argon stream through a narrow capillary tube inserted in the slurry medium. This allows samples to be directly prepared in autosampler cups. Homogenization requires a "bubbling time" with the Ar stream of only 30 s and provides a precision of about 6% as RSD in the determination of Cu, Co, Cr, Mn, Fe and Ni in glass [43]. (e) Predigestion of the slurry. This procedure can facilitate extraction of the target analyte into the liquid phase. In contrast to the extensive sample pretreatment inherent in the traditional fusion and digestion procedures, predigestion of a slurry only requires partial decomposition of the sample, so it is not time-consuming. For example, partial wet oxidation of biological and plant materials can be accomplished by using concentrated sulphuric acid with subsequent analysis of the carbonaceous slurry formed, all with increased expeditiousness and reliability relative to dry ashing and extensive wet oxidation. The difficulties of stabilizing slurries containing large particles in the analysis of sediments and particulate matter can be overcome by predigestion (viz. by adding a small amount of concentrated nitric acid to the sampling cups containing the slurry), which results in partial extraction of the analytes into the liquid portion of the slurry. Metals in glass can be predigested in the slurry by adding a low concentration (3%) of hydrofluoric acid and homogenizing by gas mixing. The predigestion step can be implemented either before or after loading the cup with sample. (f) Electric dispersion in a condensed medium. The electric dispersion of metallic samples in a condensed medium produces a colloidal dispersion that is suitable for slurry preparation as judged by the characteristics of the sols formed upon spark ablation. The suspension contains metallic particles of size less than 1 lam and is highly stable. In this way, slurries of metallic materials can be prepared and the need to machine them for powdering avoided, which reduces contamination risks. The spark ablation can be done in both aqueous and organic solvents. (g) Laser ablation, which is discussed in Chapter 9. The particle size of the solid material used to prepare the slurry can influence its stability, deposition and atomization efficiency which, in turn, can affect accuracy and precision. Various types of equipment for grinding solids have been used to prepare slurries for ETA-AAS including mixer mills, other ball mills, and bottle and bead mills. The appropriate particle size varies among materials depending on various parameters such as the homogeneity of the original material with respect to the analyte, the amount of sample used for analysis and the particular diluent employed. Particle size can also influence the homogeneity of the slurry through the rate of particle sedimentation and Particle size

362


the amount of analyte extracted into the liquid phase. When large particles are present in the slurry, problems may also occur due to discrimination of the larger particles by the autosampler and irreproducible injection of the sample into the atomizer. Notwithstanding the decisive importance of particle size in the materials used to prepare slurries, information about particle size distribution is often scant or unreported in publications dealing with slurry sampling. Also, when particle size is reported, the sample has usually been ground to pass through a given mesh dimension and only the maximum particle size of the material is stated. Wibetoe et al. [44] studied the potential of using a Coulter particle analyser with a view to developing a method for measuring the effect of grinding of solid materials prepared for slurry sampling. This method would allow particle size distributions to be plotted and/or quantified in various ways including number-size and volume-size plots, size being expressed as a volume, surface area or (most often) diameter. Plotting volumes of particles against size is advantageous as detailed information about the larger particles is lost when the distribution is reported in terms of number-percentage. Plant material (viz. flowers, leaves, the stem and roots from the sample plant) was used to demonstrate the approach. Precision was found to increase with decreasing particle size, particularly in the determination of Ni and Cu; also, accuracy increased with increasing grinding time (see Table 8.1). Rather low concentrations were obtained with 1 min grinding for all plant parts and the three metals. Inaccuracy was probably the result not only of the poor precision achieved, but also of all the systematic errors probably present (all results were too low). From the data in Table 8.1 it follows that grinding for as long as 60 min was not always necessary for this type of material as the precision and accuracy obtained after 5 min of grinding was more than adequate in many cases. One other important factor in preparing a slurry is its concentration. Samples with a high analyte content are easier to analyse with the slurry technique than by direct solid sampling as the slurry can be readily diluted in the former. However, the slurry can only be diluted to a certain extent: precision degrades with a high dilution as a result of only a small number of particles remaining in the slurry. On the other hand, when the analyte content in the original sample is very low, the concentration of the slurry can be increased accordingly the pipetting efficiency, however, can deteriorate with more concentrated slurries. One other factor to be taken into account is that increasing the slurry concentration can enhance matrix effects. In food analyses, the RSD is typically less than 4% when using particles less than 50 lam in d i a m e t e r - provided the slurry concentration is below 10% m/v. Accuracy deteriorates when the slurry concentration exceeds 5% as a result of the matrix effects. Slurry concentrations above 5% result in inefficient deposition of the slurry aliquot [41]. The errors associated with slurry sampling have been characterized and found to arise from uncertainty in the sample volume and the number of particles it contains, as well as from variations in the mass of individual particles. These errors can be minimized by using small particles, concentrated slurries and narrow particle size distributions [45]. Some authors [46] claim that using slurry nebulization is only advisable when no efficient alternative digestion procedure is available. The dilution factor of the sample in slurry nebulization is at least as high as in normal solution nebulization. The concentrations

Slurry concentration

TABLE 8.1 RESULTS O F THE ULTRASONIC SLURRY SAMPLING ETA-AAS DETERMINATION O F Co, Ni AND Cu IN PLANT PARTS AND OLEA LEAVES CRM, WITH INCREASING GRINDING TIMES; N = 5 (DIFFERENT SLURRY PREPARATIONS). THE CERTIFIED OR INDICATIVE VALUE FOR THE CRM IS GIVEN IN BRACKETS ~

~~

Slurry sampling grinding time 1 rnin

Mean, Pgk CO Flowers Leaves Stem Roots Olea Leaves CRM

3.5 5.8 2.2 4.4 0.17 (0.2'"1)

5 min RSD, %

18.1 19.7 21.0 20.6 22.2

Mean, Pgk 6.5 9.2 5.7 8.3 0.18

Decomposed samples's"

60 min RSD. Yo

Mean, Fgk

RSD, YO

Mean, Pdg

RSD,

13.1 13.2 15.1 17.5 19.1

6.7 11 4.8 8.5 0.19

3.6 7.7 3.0 3.5 16.7

6.0 10.0 5.3 8.7 0.17

7.0 11.5 11.5 2.6 11.8

8.7 13.2 7.0 11.7 10.2

65 75 21 64 8. I

6.0 10.3 4.8 2.3 6.3

60 73 21 65 7.5

7.2 7.3 13.3 10.6 16

O h

Ni Flowers Leaves Stem Roots Olea Leaves CRM Clf Flowers Leaves Stem Roots Olea Leaves CRM

43 50 12 37 3.8 ( 8 9 52 112 43 265 44 (46.6*1.8'"')

18.8 15.4 15.2 15.2 19.3 15.0 13.3 17.4 13.4 14.1

63 77 20 63 7.3 I I5 188 125 40 1 45

12.0 11.4 10.6 10.4 12.3

120 190 I30 395 46

8.7 7.5 6.3 4.9 6.7

102 195 140 408 46

9.0 5.1 10.7 1.3 4.6

n = 6 ; I h i 45 min grinding for CRM; It' value given on CRM certificate (not certified value); ILIicertified value (mean f 95% confidcnce interval of the mcan) Reproduced with permission of Springer-Verlag

(,Ii

364


of dissolved solids in analytical solutions are even higher as the dispersing agents alone account for 0.5 to 1%. In addition, contamination due to impurities in the grinding agents may result in analytical errors. Polyatomic ions formed from the components of the grinding agents or the dispersants can also cause interferences. 8.2.4. Steps of an electrothermal solid sampling process

In addition to sample-related aspects, spectrometer control functions must be programmed to occur at specified times within the graphite furnace programme. While the number of steps within each programme is variable, 6 steps constitute the typical ETA programme for slurry samples (similar to that for liquid samples), which reduces to 5 steps when the solid sample is introduced as such and the drying step is thus omitted. The operational sequence thus involves (1) drying, (2) pyrolysis or ashing, (3) cooling down (optional), (4) atomization, (5) clean-out and (6) cooling down. Figure 8.4 shows a typical temperature programme, the steps of which are discussed below. Do,ing

Once the slurry has been driven to the furnace using one of the procedures described in the previous section, it must be dried at a low enough temperature to avoid sample splattering, which would result in degraded analytical precision. The temperature to be reached depends on the nature of the liquid phase in which the solid is dispersed. Temperatures around 100-120~ are common for aqueous solutions, but a value just above the boiling point of the dispersant is advisable. Use of a temperature "ramp" provides a variable time over which the temperature is raised. An extended ramp time provides a slower, more gentle increase in heating. When a platform is used, the temperature lag of the platform relative to the tube walls provides a natural ramping effect. Longer times are used when the sample is to be atomized from the tube walls. After the temperature ramp, the furnace is held at the selected drying temperature until drying is complete. Since the volume of slurry used is typically of only a few microlitres, the drying hold time is usually less than 1 min (usually 1-3 seconds per microlitre of dispersant). The pyrolytic platform invariably requires the use of somewhat higher temperatures than those needed for sampling from the tube walls. During the drying process, the internal gas flow is normally kept at its default maximum value (250-300 ml/min) to purge the vaporized solvent from the tube. Pyrolysis or ashing

The purpose of this step is to volatilize inorganic and organic matrix components selectively from the sample, leaving the analyte element in a less complex matrix for analysis; as a result, the duration of this step is a function of the complexity of the sample and can vary between 10 and 30 s. During it, the temperature is increased as rapidly as possible to volatilize matrix components but is kept below the level at which analyte losses might occur. The temperature to be used in the pyrolysis step will depend on the particular analyte and matrix. Recommended temperatures are usually provided in the

Devices/or treatment prior to introduction into atomic spectrometeJw 2500

-

2000

-

1500

-

1000

-

500

-

365

RAMP

,,

v

DRY

PYROLYZE

J

',--,,--, ATOMIZE

TIME

Fig. 8.4. Typical temperature programme for an electrothermal atomizer (ETA).

documentation accompanying each commercially available graphite furnace. The internal gas flow is again kept at 250-300 ml/min in order to allow volatilized matrix materials to be driven off. With some samples, it is advantageous to change the internal gas (e.g. to air or oxygen) during the pyrolysis step to facilitate sample decomposition. Pre-atomization cool-down

This optional step is used mainly with longitudinally heated furnaces in order to cool the furnace prior to atomization as the heating rate is a function of the temperature range to be encompassed. The rate of heating increases as the temperature range is broadened. The use of a cool-down step prior to atomization maximizes the heating rate and extends the isothermal zone within the tube immediately after heating. The extended isothermal zone has been shown to improve sensitivity and reduce peak tailing for a number of elements including those which are typically difficult to atomize in the graphite furnace. This step is not required with transversally heated furnaces as the isothermal zone extends the length of the tube in this type of system. Atomization

In order to produce an atomic vapour of the analyte elements, the temperature of this step is increased to the point where dissociation of volatilized molecular species occurs.

366


The atomization temperature is a function of the analyte element and must be increased as quickly as possible. Therefore, ramp times will normally be set to a minimum value resulting in the highest possible heating rate. It is also desirable to reduce or, preferably, completely stop the internal gas flow during atomization in order to extend the residence time of the atomic vapour in the furnace, thus maximizing sensitivity and reducing some interferences. It is worth noting that the analytical signal does not increase linearly with increase in the atomization temperature because the rate of loss of atomic vapour from the atomizer also increases with increasing temperature. Clean-out and cool-down

After atomization, the furnace may be heated to still higher temperatures to burn off any sample residue remaining in it. An optional cool-down step then allows the furnace to return to a near-ambient temperature before the next sample is introduced.

8.2.5. Instrument parameters affecting solid sampling with electrothermal atomizers and vaporizers As stated above, the sample dispensed into the furnace is subjected to a multi-step temperature programme. When the temperature is increased to the point where sample atomization or vaporization occurs, measurements are made directly or after transporting the vapour to the ICP torch or MS instrument. The variables under operator control in this situation are as follows: (1) (2) (3) (4)

Temperature (viz. the final level); Ramp time (viz. the time for temperature rise); Hold time (viz. the time during which the final temperature is maintained); Internal gas (viz. its type and flow-rate).

The value of each of these instrument parameters to be used in each step depends on the instrument itself, the sample matrix and the analyte.

8.2.6. Use of modifiers in electrothermal solid sampling The concept of chemical modification (CM) is extremely popular in modern electrothermal-assisted atomic techniques. As per IUPAC's recommendations [47], "in order to influence processes taking place in the atomizer in the desired way, reagents called 'chemical modifiers' may be added. These can help to retain the analyte to higher temperatures during pyrolysis, remove unwanted concomitants or improve atomization in other ways". However, there is a tendency towards broadening the scope of this term, starting from the classical and still used term "matrix modifier", "matrix/analyte modifier" or "instrumental matrix modification" to indicate the useful effects of the type, pressure and flow-rate of protective gas or gas mixtures; "internal matrix modifier" for matrix constituent(s) with favourable effects on processes in the atomizer, either by themselves (e.g. refractory components) or upon addition of suitable promoters; "permanent modifiers" for


367

graphite surfaces, which are coated with high-boiling point noble metals or carbides. A simplistic current classification based on the duration of their effect divides modifiers into two categories: conventional and permanent. Conventional modifiers

Conventional modifiers must be added to the atomizer prior to every atomization heating cycle because they are expelled during the atomization and/or clean-out steps. Some research into transient modifiers is currently being devoted to their mechanisms of action; such is the case with the studies on the film produced by the decomposition of ascorbic acid on the platform surface, which absorbs Au atoms preferentially [48], or the use of synchrotron X-ray absorption spectroscopy to further elucidate the mechanism of Pd modification of selenium [49]. However, a number of publications have been devoted to the applications of conventional modifiers over the past two decades, but particularly during the 1990s. Specially prominent in this context are the studies of Burguera et al. [50] on the addition of modifiers to Massmann-type furnaces for the determination of Cr in urine, which led to increased background absorbance signals which is generally not the case with THGAs, however. Also worth noting is the study of the effectiveness of Pd and phosphate modifiers in a tungsten furnace, where both modifiers were found to increase the maximum permissible pyrolysis temperature for Cd but not to the extent achieved in a graphite furnace [51]. Permanent modifiers"

Permanent modifiers are a result of the search for a solution to the high blanks caused by repeatedly adding transient or conventional modifiers as even the highest purity salts and compounds of the modifiers may contain measurable concentrations of metals. Unsurprisingly, interest in the use of permanent modifiers has risen in recent years. A permanent modifier interacts strongly enough with the graphite furnace to be retained through multiple firing cycles, and any trace metal impurities are usually removed during its initial thermal conditioning. Permanent modifiers can also increase the lifetime of the graphite furnace. Existing chemical modifiers span all aggregation states and a wide variety of chemical families, namely: (a) Inorganic salts the most popular group the modifiers being both cations such as Pd(II), Ni(ll) or N H z , and anions such as nitrate, phosphate, vanadate or tungstate. Nitrate and ammonium ions, respectively, have proved the best counterions for these salts. (b) Organoelements and complexed forms of modifiers, which are useful when samples or analytes are transferred into organic phases such as extracts, leachates, oils, fats, petroleum products, organometal [lead acetate or acetylacetonate] solutions, etc. (c) Inorganic acids (HNO 3, H3PO4) and bases (aqueous ammonia). (d) Organic acids (acetic, tartaric) and bases (e.g. solvents for biological tissues including TMAH, TAAH and thyamine hydroxide).

368


(e) Complexing agents such as EDTA, citric acid or citrate ion for keeping the analyte,

modifier and/or matrix components in solution.

(f) Reductants for conditioning noble metal modifiers (e.g. ascorbic acid, citric acid, NH2OH/HCI, H2-Ar). (g) Oxidants [HNO 3, Mg(NO3)2, O2, air alternate gases] for promoting in situ ashing of organic matrices. (h) Reactive alternate gases (H,, O~, freons). (i) Miscellaneous organic additives with complexing action (e.g. organic acids and solvents, surfactants). _

_

Obviously, some of these single-component modifiers may play more than one role. In addition, the efficiency of chemical modifiers can be improved in various ways, namely: (a) Palladium and other noble metals often perform better in the presence of reductants, resulting in a "reduced Pd modifier". Common reductants include ascorbic, oxalic and citric acids, hydroxylamine hydrochloride and hydrazine sulphate. Thermal conditioning of noble metals by injection and pretreatment at 1000~ followed by cool-down and then sample insertion are also widely used. Pre-reduction of noble metals (Ga, In and T1 mainly) has proved especially important with some difficult analytes forming strong bonds with chloride and other halides. (b) Metals other than Pd (Ir, Pt, Rck Ru) may perform better in special analytical situations by virtue of their lower volatility. (c) Mixed noble metal modifiers may be preferred on account of their lower volatility and higher vaporization temperature. (d) Mixed modifiers containing noble metals and oxide- or carbide-forming elements are frequently not only more efficient but also more universally applicable to larger numbers of analytes. (e) (Noble) metal modifiers often exhibit improved efficiency when applied on carbidecoated graphite tubes or platforms, an effect similar to, yet lower in magnitude, than that observed in the presence of their corresponding mixed modifiers. (f) Permanent modifiers such as carbide coatings (e.g. NbC, TaC, WC, ZrC), less volatile noble metals (Ir, Rh) or noble metals on carbide coatings are becoming increasingly popular. The most salient effects sought in modifiers are as follows: (a) Efficient thermal stabilization of low to moderate volatile analytes (over 30 elements including such important analytes as Bi, As, Cd, P, Pb, Sb, Se, TI and Sn), particularly in matrices of volatility comparable to that of the analyte. (b) Isoformation of various analyte species such as arsenite, arsenate, monomethyl arsonate, arsenobetaine, arsenocholine, etc., with a view to levelling off their integrated absorbance by transformation into less volatile chemical forms. (c) Increasing the volatility of unwanted contaminants, which facilitates their removal during the pyrolysis step (e.g. elimination of the bulk chloride matrix in analyses of seawater or urine by adding NH4NO~, dilute HNO 3, ammonium salts of organic additives or H2-Ar as alternate gas during pyrolysis.

Devices ~br treatment prior to introduction into atomic ,wectrometers

369

(d) Facilitating in situ ashing rather than charring of organic matrices using air or 0 2 alternate gas during the (low-temperature pyrolysis step). (e) Thermal stabilization of some interfering species such a s P2 or PO in phosphate-rich samples (e.g. bone, biological fluids) upon addition of noble metals or Ni so as to delay their vaporization during the atomization step (as P atoms rather than molecules). (f) Transformation of some interfering compounds into less harmful species (e.g. binding sulphate by mixed modifiers containing salts of alkaline-earth metals such as Pd-Mg-Ba or Pd-Sr). (g) Modifier action as volatilizers (i.e. by facilitating low-temperature atomization of the analyte before the bulk matrix). (h) Ensuring better contact between the sample and atomization surface by adding wetting agents and/or organic reagents for smooth drying and/or pyrolysis. (i) Enabling in situ speciation analysis, as in the fraction vaporization of Se(IV) alone as a volatile piazselenol complex during the pyrolysis step, Se(VI) remaining in the atomizer for selective quantitation. (j) Increasing sensitivity. While very attractive, this aim and (eventually) result of chemical modification may just reflect the situation that conditions for efficient atomization cannot be obtained in the absence of modifiers in many analytical cases (e.g. in the determination of high-volatile organotins, where premature losses can be avoided or greatly reduced in the presence of an organopalladium modifier). (k) Trapping volatile hydrides in hydride generation prior to electrothermal atomization by using graphite surfaces modified with carbides, noble metals or both. (l) Improved resistance of some Ta, W and Nb carbide-coated surfaces to attack and permeation by acid digests, organic solvents and extracts, corrosive matrices and modifiers, among others. (m) Providing better conditions for simultaneous multi-element assays through thermal stabilization of volatile analytes and employing more uniform programmes for groups of several analytes (e.g. As and Se; Cd, Cr, Pb and Ni). The following are a few selected examples of the use of both conventional and permanent chemical modifiers of diverse nature and causing different effects. (a) The use of a reactive gas such as Freon 1,2 as a halogenating reagent to improve the release of analytes such as A1, Ag, As, Bi, Ca, Co, Cr, Cu, Fe, Ga, K, Li, Mg, Na, Ni and Pb in graphite and silicon carbide for their determination by ICP-AES [52]. The chlorinated compounds of these elements are much more volatile than the elemental species themselves. By this means, the vaporization temperature can be considerably reduced, the release of analytes from the samples improved and, in some instances, the reaction of carbide-forming elements with the furnace diminished. This should lead to improved transport efficiency and hence to increased sensitivity. Figure 8.5 shows the emission signals obtained for Li with and without the addition of Freon in processing an aqueous standard solution and a graphite sample. Polytetrafluoroethylene (PTFE) has also been used in slurry introduction sampling (SIS) for fluorination (and efficient volatilization) of cadmium from solid biological materials [53,54], and of Co and Ni from silicon dioxide powder [55].

370

Devices Jbr treatment prior to introduction into atomic ,wectrometers

A) 700 -

AQUEOUS STANDARD SOLUTION

600 -

2.5 ng Li WITH FREON A int= 490 mV

500 %" E

400 300 200 -

5 ng Li WITHOUT FREON

'00-V

0

" ~ - V v v -'VVIVV'V~IM" V"

2

4

6

I

I

8

10

v-.

~rvvv.v~A~W~

12

/

14

TIME, s

B)

GRAPHITE SAMPLE

250 -

Li in 4.9 mg GRAPHITE WITH FREON A int = 184 mV

200 -

150E

Li in 5.5 mg GRAPHITE WITHOUTFREON

100]

0

4

/

8

12

14

TIME, s

Fig. 8.5. Emission signals for Li as obtained with and without the use of Freon as additive. (A) Aqueous standard solution. (B) Graphite sample. (Reproduced with permission of the American Chemical Society.)

Devices]or treatment prior to introduction into atomic spectrometers

371

TABLE 8.2 DETERMINATION OF ARSENIC IN SEDIMENT AND SOIL SAMPLES BY ETA-AAS (n = 5). PERMANENT MODIFIER, 250 lag OF W + 200 lag OF Rh; CONVENTIONAL MODIFIER, 5 lag OF Pd + 3 lag OF Mg(NO0~. CONFIDENCE INTERVAL AT 95% CONFIDENCE LEVEL (ts,,d~.,1, = 2.776) As, lag/g Slurry Sample

Buffalo River sediment SRM 2704 Pond sediment NIES 2 Lake sediment SL-I River sediment CRM 320 Soil-5 IAEA Soil-7 IAEN"' Sediment I Sediment II

Certified value

W + Rh

Pd + Mg

Digested Pd + Mg

23.4 + 0.8 12.0 + 2 27.5 + 2.9 76.7 + 3.4 93.9 + 7.5 13.3 ---

23.3 + 0.3 12.1 + 0.4 26.9 + 1.0 78.2 + 1.3 90.4 + 2.5 13.7 + 0.5 65.9 + 2.4 44.2 + 1.2

23.6 + 0.4 11.8 + 0.5 28.7 + 1.2 75.2 + 1.8 92.7 + 3.6 13.1 + 0.7 66.8 + 3.0 46.1 + 2.2

22.8 + 0.5 13.1 • 0.8 26.4 + 1.6 79.1 + 2.2 89.4 + 4.4 13.8 + 1.4 67.4 + 4.0 45.4 + 3.8

~"~Confidence interval: 12.5-14.2 lag/g Reproduced with permission of the Royal Society of Chemistry

(b) A recent study on the use of W - R h permanent modifiers in ETA-AAS for the determination of lead in slurries of biological materials revealed that the modifier remained stable over approximately 300 analytical measurements when a volume of 20 ~1 of slurry containing up to 1.5% m/v was delivered to the atomizer; the permanent modifier increased the tube lifetime by approximately 100% relative to untreated integrated platforms. Also, the sensitivity decrease during the atomizer lifetime was less marked than with conventional modifiers, which resulted in increased sample throughput. The atomizer lifetime was restricted to the THGA wall durability; in fact, the W - R h treated platform remained intact after more than 650 analytical firings. The results thus obtained in the determination of lead were consistent with those provided by digested solutions and the use of Pd/Mg(NO3) 2 [56]. (c) A comparison of the use of a W - R h permanent modifier and a Pd/Mg(NO~) 2 conventional modifier for the determination of As after slurry introduction sampling of sediments into ETA-AAS equipment revealed the results of both approaches to be consistent (see Table 8.2) [57]. (d) Lead as a modifier has been used to improve the determination of various analytes in highly different matrices and also using different detectors. Thus, the presence of palladium and ammonium nitrate in the determination of lead in fish by slurry sampling and ETA-AAS substantially improved the signal by increasing the atomization temperature and reducing differences in analyte transport from the sample and calibration solutions

372


[58]. The use of Pd and NaCI was found to have a similar effect on the determination of As, Mn, Pb and Se in coal by slurry introduction sampling (SIS)-ETA-ICP-MS [59]. Despite their advantages, modifiers are subject to a number of side effects and restrictions that are increasingly better known. While some of their problems can be reduced, others can only be avoided by using an alternative modifier. Their most serious shortcomings are as follows: (a) Impaired effectiveness with real matrices owing to competitive side reactions with, or inhibition by, matrix and/or solvent constituents. Pyrolysis temperatures often happen to be lower than those established with simple aqueous solutions. (b) The need for longer temperature programmes, which arises from the unfavourable kinetics of the solid-phase reactions by which analytes are stabilized and matrix interferences avoided. (c) High reagent blanks produced by large amounts of modifier, which can lead to the need for unacceptably cumbersome blank corrections. This shortcoming can be circumvented by using high-purity and thus expensive reagents or additional purification steps involving ion-exchange or liquid extraction. (d) Overstabilizing effects resulting from the use of large amounts of modifier, extended pyrolysis times at high pyrolysis temperatures, gradual accumulation of non-volatile modifiers or migration of the modifier to the cooler ends of a longitudinally heated atomizer. (e) Corrosive action on graphite and pyrocoatings, which has a strong effect on the atomizer lifetime. ~f) Irreversible contamination of graphite parts and their surrounding areas by Cr, Mg, Mo, Ni, Pd or V modifiers, which precludes their subsequent determination as analytes. (g) High background absorption from the modifier itself, which requires effective correction. (h) High toxicity or other harmful effects. (i) Compatibility problems (some modifiers must be stabilized with high acid concentrations in order to avoid hydrolysis and their disappearance from the solution). 0) Restrictions derived from the lack of non-empirical information and markedly discrepant results. Permanent modifiers are subject to specific additional shortcomings such as the following: (a) Partial, non-uniform coating of platform and tube surfaces, which produces "islands" that act as atomization sites; (b) Relatively short lifetimes; (c) Restrictions upon temperature programmes; (d) Limited applicability to fairly complex solutions; (e) Overstabilization of some carbide-forming and less volatile analytes; (f) A lack of interest among equipment manufacturers.

Devices Jot treatment prior to introduction into atomic spectrometers

373

Modifiers are essential for many ET-AAS applications. The new generation, permanent modifiers, is receiving increasing attention by researchers, which will help to overcome their present drawbacks.

8.2.7. Shortcomings of electrothermal sampling The direct introduction of solids into electrothermal atomizers and vaporizers poses a series of problems especially prominent among which are the following: (a) Inserting a solid sample into an electrothermal device is not as easy as introducing a liquid sample; in fact, direct insertion of a solid sample requires extreme care and training. Some loss or contamination can easily occur during weighing and/or transfer from the balance to the atomizer. This problem is less critical with slurry sampling. (b) In contrast to liquid samples, which are inserted in a direct manner, a separate weighing operation is required for each replicate. This can make the analytical procedure time-consuming to an unacceptable extent for routine determinations. Slurries allow several determinations to be performed with a single sample preparation. The automation of the solid sample insertion operation has resulted in some improvement in routine determinations. (c) Standardization poses problems not encountered with liquid samples. Matrix dependence of peak shape and incomplete release of the analyte from the solid sample are two common occurrences. Calibration with aqueous standards is not always possible and the method of standard additions fails when the added analyte is not uniformly affected by the matrix. Calibration with certified reference materials with matrices highly similar to those of the samples is the most reliable approach in this context. (d) Chemical modifiers are less effective for the direct atomization of solids as they cannot interact in an effective manner with the analyte, which is occluded within the sample particles. Various chemical modifiers have been tested with a view to improving the interaction. (e) Solid samples are more difficult to dilute when the analyte is highly concentrated as this entails lowering the instrument sensitivity or previously diluting the sample with an appropriate solid buffer. The slurry sampling technique affords dilution but requires extreme care to avoid errors (e.g. when only a few solid particles remain in the slurry). (f) Precision is worse than with liquid samples. Relative standard deviations are typically in the region of 10% but can be improved by either decreasing particle size or increasing the amount of sample used for the determination when sample inhomogeneity is the most influential factor. With many certified reference materials (CRMs), sample inhomogeneity must be considered for amounts of sample below 200 rag, which is much larger than the 0.1-10 mg typically used in solid sampling. (g) Parts of the matrix can remain in the furnace after atomization and damage the atomizer. Residual matrix build-up can also block the light beam emitted by the radiation source and affect analytical performance. This shortcoming can be circumvented by using a clean-out step after each series of measurements.

374


(h) Spectral interferences are more pronounced when the solid sample is atomized in a direct manner. High and structured background signals can cause large errors. The use of a powerful background correction system is thus required if accurate results are to be obtained. Oxygen ashing can also help to remove non-specific absorbance interferences. (i) Slurry introduction is subject to specific problems arising from the need to maintain a stable, homogeneous slurry. The determination of refractory metals is hampered by incomplete release during (J) atomization and by occlusion in the solid matrix. High atomization temperatures relative to liquid samples are required under these conditions to release the analyte within a reasonable time. (k) Preparing blanks can be cumbersome, particularly with ground samples. Many of the previous problems have been partly solved with the implementation of new instrument developments and improvements in analytical methodology over the past two decades. Progress in three different lines (viz. the design of graphite atomizers specially adapted for handling solid samples, the automation of solid sample insertion and the use of stable-temperature platform furnaces) has helped overcome some of the drawbacks of solid sampling with electrothermal atomizers and vaporizers. 8.2.8. Calibration in methods involving electrothermal sampling of solid samples and slurries

Notwithstanding their selectivity, atomic techniques are largely used for quantitative rather than qualitative analysis. Some theoretical evaluations have been made with a view to using solid sampling ETA-AAS for screening purposes by simulating more than 18 000 results corresponding to different possible situations and analysing them using information theory. In the light of the results, the median can be used to better effect than the mean value as the former is less markedly affected by the potential presence of outliers. Using the median and typical working conditions, the minimum number of measurements to be made in order to ensure a recall of over 0.95 in a screening method ranges from 5 to 20, which involves between 15 and 60 minutes' work [60]. The solid matrix can strongly influence the shape of the signal provided by the atomic spectrometer. Such influence hinders the use of the peak height for the target analyte. The mechanisms governing the processes occurring in atomizers have been modelled with a view to minimizing or suppressing the effect of the solid matrix [61-64]. The fast transient signal calls for a fast instrumental response, which should preferably be integrated. Linearization algorithms allow results to be improved and the linear range to be extended [65]. The use of external standards (e.g. Ar ~) in ICP-MS) to compensate for variations in mass sampled and analysed is a usual practice for correcting matrix-induced signal suppression effects [66]. Calibration can be done using various approaches involving CRMs, synthetic solid standards, the method of standard additions or aqueous standards. The procedure involving CRMs requires that a reference material containing an appropriate concentration of the target analyte be available. This is not a general calibration


375

method owing to the lack of reference materials with certified analyte concentrations spanning the range of interest (e.g. in environmental samples). In any case, calibration with CRMs has been used with metal-containing solid samples [67], sewage sludge [68], biological samples [69] and plant material [70]. One substantial disadvantage of reference materials used as standards is that the certified concentration usually has an uncertainty that is high compared to the uncertainty of the method. Consequently, the use of CRMs as calibrants introduces high, frequently ignored uncertainty in the final value. In some instances, when CRMs are not available, a synthetic solid sample matching the composition of the original sample can be used instead. Obtaining synthetic solid standards is a difficult, time-consuming process that is only justified when both the number of samples and that of analyses is high (e.g. in the steel industry). The method of standard additions has been used to suppress the influence of the bulk composition of solid samples. It requires that the analyte contained in the solid sample and the added analyte be identically affected by the matrix. This method can be used in three different ways in connection with solid samples. One involves adding increasing amounts of analyte to fixed amounts of sample. In the second, a single amount of sample is subjected to a single addition of analyte from a standard solution, which can lead to inaccuracy in the final result. In the third approach, both the amount of solid sample used and that of standard added are varied; the instrumental signal is a function of two independent variables and can be extracted by multiple linear regression. The use of aqueous standards entails assuming that the instrumental signal will be independent of the bulk matrix and dependent on the analyte concentration alone. This requirement is usually not met because of both spectral and chemical interferences from the matrix. For calibration with aqueous standards to be successful, a series of instrumental requirements including expeditious signal processing, the use of integrated measurements, appropriate background correction and atomization under isothermal conditions must be met. With solid samples, absolute calibration of the autosampler or pipette used to dispense the standard solutions is also important because the accuracy of the determinations is directly dependent on the accuracy of the pipette. A comparison of the use of CRMs and multi-element solution standards revealed that the results of both approaches were similar, with not much influence of the residual organic matrix on the transport efficiency. Adding dilute nitric acid to the solid sample inside the graphite boat of the furnace allowed removal of most of the organic matrix during the pyrolysis step. Consequently, calibration against aqueous standard solutions provided analytical results that were as good as those obtained with the method of standard additions [71]. A comparison of three calibration methods based on the use of CRMs, generalized simplified standard additions and multiple standard additions revealed the last to be the most practical and reliable [72]. The use of solid standards introduces a different concept of calibration. Traditional calibration is done by changing the analyte content. With micro-weighings, accurately replicating the same sample mass is difficult. Therefore, calibration with solid standards for ETA is done with powdered reference materials by varying the sample mass. Conventional two-dimensional calibration focuses on the analyte mass without considering the corresponding matrix mass. The analyte mass is an analytical quantity derived


376

O n Zk O

GBM CRM GBW CRM

07309 320 07305 280

'b

~b

.

v,lt..

-

,. 'k~

"~ ~

.t-,,~b

~" e0,.,., ~

_ @'v

Fig. 8.6. Three-dimensional plot for Zn illustrating different calibration methods. Traditional calibration by variation of the analyte content is shown using four different samples. Calibration by variation of the sample weight is shown using the certified reference material GBW 07305. (Reproduced with permission of Springer-Verlag.)

TABLE 8.3 RESULTS OF THE DETERMINATION OF Pb USING SOLID SAMPLING G F - A A S

CRM/RM W-I BM Mica-Mg Mica-Fe TB GBW 083802

Certified value, ~tg/g

Analysed value '"1 3D, lag/g

Recovery %

Analysed value ~ 2D, lag/g

Recovery %

7.5 + 1.5 13 + 2.0 13 + 2.0 9+3 13 + 3 8 + 1.4 14.2+2.7

7.1 15.0 8.7 11.1 6.4 13.5

95 106 97 85 80 95

6.8 14.8 8.5 10.6 6.3 12.9

91 114 94 82 79 91

• + + + + +

~"' + standard deviation (n = 5) Reproduced with permission of Springer-Verlag

1.6 1.9 1.6 0.6 0.7 1.1

+ + + + + +

1.8 1.9 1.8 1.3 0.5 1.3


377

from the sample weight and analyte content. The disadvantage is that, in materials with a strong matrix influence, the matrix mass is not taken into account. In order to include the sample weight and analyte content in the calibration, the transition to three-dimensional calibration is required. Figure 8.6 shows a 3D plot for Zn that illustrates the different approaches to calibration in this context, namely: traditional calibration by changing the analyte content or by varying the amount of sample. Table 8.3 summarizes the results provided by the 2D and 3D calibration methods. As can be seen, recovery and precision are better with 3D calibration. Confidence intervals for the 3D calibration method have recently been modelled [73]. 8.2.9. Applications of electrothermal solid sampling prior to introduction into an AAS, ICP-AES, AFS or ICP-MS instrument

The number of applications of atomic techniques based on solid or slurry sampling is so large that only a comparatively minute fraction is discussed in this section. Interested readers are referred to the biannual reviews of Analytical Chemistry and the atomic spectroscopy update in the Journal of Analytical Atomic Spectrometry, among other sources, for more extensive information. A specific review of the uses of graphite atomizers modified with high-melting carbides has been published by Volynsky that includes virtually all metals determined in this manner [74]. As with liquids, the four atomic techniques have complementary uses with solid samples. When axial inductively coupled plasma atomic emission spectroscopy (ICPAES) hit the market in the early 1990s, some manufacturers of graphite furnace atomic absorption (GF-AAS) systems feared it was time to start looking for a new job. Despite the increased pressure from ICP-based and other competing techniques, however, GF-AAS continues to hold a place in the elemental analysis market [75]. Considerable efforts have been made over the last decade to combine solid sample insertion methods with ICP-MS. Many systems which had stood the test with ICP-AES were adopted for use with ICP-MS. Based on its excellent multi-element determination capabilities, its high sensitivity and its ability to handle transient signals, ICP-MS is regarded as a more suitable combination with solid sampling devices than is ICP-AES; however, there has been a marked decline in work concerned with the direct introduction of solids into ICP instruments in the last few years. Below are described selected examples of the use of the four techniques following electrothermal pretreatment. Atomic absorption spectrometry was the first in its time and perhaps the most widely used with solid samples. There are very few references to the use of flame as atomizer in handling solid samples in combination with an air-acetylene flame [76] or slurry sampling [77,78] prior to AAS. When the concentration of the target analytes is high enough (e.g. in the determination of Cd, Pb, Hg, Sb and As in soil, rock and sediment samples), both the sensitivity and the accuracy can be improved by dilution with graphite powder [79]. The determination of other metals in high-purity titanium using a calibration graph obtained with standard solutions provides limits of detection ranging from 0.02 (Mg) to 30 ng/g (Sn) [80]; those obtained in high-purity tungsten range from 0.01 (Mg) to 1.7 ng/g

378

Devices for treatment prior to introduction into atomic spectrometers

(Zn), so they are far better than the limits typically provided by digested samples [81]. In dealing with slurries, very long shaking times (1-4 h) with ultrasonic vibrators are most often required [82,83]. Some prefer to use ultrasonic assistance to leach solid samples in the autosampler cups (using a small ultrasonic probe) [84-86]. Other methods such as that for the determination of Ag are special; following preconcentration on a chelate resin, the solid is suspended in 1 ml of dilute HNO 3 and inserted into the graphite furnace [87]. The determination of total mercury in natural gas liquid and condensate by carbon adsorption and slurry sampling [88] is an unusual application of GF-AAS. The speciation of mercury in human hair requires the use of both solid and liquid samples [89]. Table 8.4 compares the direct analysis of pieces of high-purity titanium for a series of metals such as A1, As, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sn and Zn using ETA-AAS following sample dissolution with the results provided by other, well-established techniques such as neutron activation analysis (both RNAA and INAA). As can be seen, the need for no sample dilution in solid sampling allows the determination of many components that cannot be determined after diluting the sample in the dissolution step. The limits of detection achieved are compared with those provided by RNAA, INAA and isotope dilution mass spectrometry (IDMS) in Table 8.5; as can be seen, the ETA-AAS technique yielded better results than all others. Figure 8.7 depicts the procedure used to treat the solid sample prior to ETA-AAS, as well as other steps of the overall analytical process [80]. Table 8.6 compares solid sampling (SOS) and slurry sampling (SIS) in the determination of trace impurities in niobium pentoxide; contents are given as mean values and standard deviations based on 6 and 5 separate determinations for SoS-AAS and the other two methods, respectively. The relative standard deviations (RSDs) of SoS-AAS, which include the inhomogeneity of the material at the milligram level, are between 4 and 10% by exception, those for Co, Mn and Na are 17-20%. Taking into account the low contents determined (between 5 ng/g for Mn and 160 n/g for Fe), the RSDs can be considered excellent and indicative of high homogeneity in the material. The RSDs for the solid sampling method are similar to those for slurry sampling; however, they are significantly lower (by a factor up to more than 2) than those for solution ETA-AAS. This can largely be ascribed to the effect of the relatively large scatter of the blank in the determination of such low analyte concentrations. The limits of detection of the methods are compared in Table 8.7 with those provided by arc-AES and ICP-AES; as can be seen, SoS-AAS per.. forms better than the other methods. The usefulness of 3D calibration for SoS-GF-AAS was recently demonstrated [90]. The earliest uses of inductively coupled plasma atomic emission spectrometry (ICPAAS) for the excitation and monitoring of the signal from solid samples provided unacceptable results (recoveries between 60% and 136%) [91] which have since been improved on [92-93], particularly with slurry sampling a currently well-established choice for solid sampling analysis. Applications span a wide range of sample types from plant to refractory materials [94-103]. Table 8.8 compares the results of ETV-ICP-AES and ETA-AAS in the direct analysis of high-purity tantalum powders for other metals; as can be seen, the combination of ICP-AES with solid sampling and ETV is a powerful tool for multi-element characterization at trace levels, which is also well suited to routine analyses when provided with automated platform handling. As shown in Table 8.9, this technique can compete with ETA-AAS plus sample digestion as regards limits of detection [104].


379

Sawing and cutting sample to 0.2-5 mg pieces

Etching with 5% HNO 3+ 2% HF, washing with h.p. water

Drying under IR-lamp in a clean-bench

Q

-13_

z:~

~-z off 0 u_

"rl O

~.

{/}

O ~?

47

Checking the blank

Inserting 2-3 mg C-powder, cleaning by heating, checking the blank

Weighing up to 30 mg sample on the platform

Weighing up to 12 mg sample on the C-layer

-•!•

ETA-AAS measurement I ~

Fig. 8.7. Procedure for the treatment of high-purity titanium samples for the determination of various metal impurities.

380


TABLE 8.4 COMPARISON OF THE RESULTS OBTAINED IN ANALYSES OF TWO TITANIUM SAMPLES BY SOLID SAMPLING ETA-AAS WITH THOSE OF SOLUTION ETA-AAS, INAA AND RNAA Content, ng/g

Element

Sample

SoSETA-AAS

SolutionETA-AAS

AI

Ti- 1 Ti-2 Ti- 1 Ti-2 Ti-I Ti-2 Ti- 1 Ti-2 Ti-2 Ti- 1 Ti-2 Ti-2 Ti-1 Ti-2 Ti-1 Ti-2 Ti- 1 Ti-2 Ti- 1 Ti-2 Ti-2 Ti- 1 Ti-2 Ti- 1 Ti-2 Ti- 1 Ti-2

19 100 + 200 15 900 + 1900 9400 + 1000 42 + 6 0.95 + 0.15 0.31 + 0.04 380 + 30 34 + 3 2700 + 200 6200 + 300 29 + 3 4500 + 600 0.13 + 0.01 < 0.1 23.8 + 2.4 0.4 + 0.1 8000 + 300 1.9 + 0.1 0.8 + 0.2 1.8 + 0.4 1200 + 100 46 + 3 < 3 83 000 • 7000 450 + 50 0.46 + 0.08 0.19 • 0.04

15 800 + 600 12 700 + 600

As Ca Co Cr Cu Fe K Mg Mn Na Ni Pb Sn Zn

INAA

RNAA

7500 + 500

420 + 10

430 + 10

2900 + 100 5600 + 1800 30 + 5 4700 + 200 < 110

1 MHz results in a pseudo-continuous plasma. The most important result of the application of a high-frequency ac potential is the self-biasing that sustains the discharge and reduces sputtering in the plasma. Consider the 2 kV peak-to-peak square wave potential V,, applied to an insulating surface (creating a GD plasma) and the resultant potential of the cathode (lib) as shown in Figs 8.11A and 8.1 I B, respectively. In the initial half-cycle, the voltage on the sample cathode goes to - 1 kV and then begins positive charging to about - 0 . 7 kV. During the next half-cycle, the applied voltage is switched to 2 kV to the positive, which produces a surface potential of + 1.3 kV. The positively charged surface during this fraction of the cycle causes the electrons in the plasma to accelerate towards the cathode (sample). The increased mobility of the plasma electrons relative to the much heavier positive ions results in fast neutralization of the surface charge during this second half-cycle such that electrode surface potential approaches zero much more rapidly than during the previous half-cycle; it reaches +0.7 kV as the next cycle starts. As the polarity of the voltage is switched, the surface voltage will reach -1.3 kV (instead o f - 1.0 kV), offsetting the voltage by -0.3 kV. As successive cycles proceed, the surface voltage on the insulator sample will be further offset in the negative direction until it reaches an equilibrium level at which the electrode virtually bears a dc-like negative voltage called the "dc bias". The dc bias potential is generally one-half the applied peak-to-peak voltage and is a function of the general electrode design and discharge operating parameters. The insulating target material is alternately bombarded by high-energy ions and low-energy electrons; however, the discharge is, for all intents and purposes, continuous with respect to most analytical measurement systems. Most importantly, the sample serves as a timeaveraged cathode and is subjected to the cathodic sputtering characteristic of GD devices. It is important to bear in mind that the electrode processes, by virtue of the differing mobilities of electrons and positively charged ions, possess a capacitive nature as this defines the dc bias, which in turn ultimately has a pronounced effect on the atomization, excitation and ionization characteristics. The above-described capacitive response of an insulating target will obviously not be present in an electrically conducting cathode. In fact, the bias potential in a non-capacitive (conducting electrode) system is zero. As with atmospheric pressure, rf plasma, the rf potential in these devices is typically coupled to the insulating material via a matching (L-C) network, which serves to maximize the power delivered to the plasma by balancing the system impedance of the rf generator to that of the discharge. As such, the capacitance of this circuitry provides the capacitive response for conducting targets. Because it is governed by the same principles as the dc discharge, this dc bias voltage induces the sputtering of the cathode (sample) material, causing the sample to atomize. Sputtered atoms (predominantly) are then available to diffuse into the negative glow region for subsequent excitation and ionization. Detection of analyte species by atomic


391

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t

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21:

31:

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f

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TIME

Fig. 8.11. (A) Establishment of a dc bias potential at an insulating surface under the influence of a high-frequency, square-wave potential. (B) The resulting potential on the cathode surface. (Reproduced with permission of the American Chemical Society.) absorption and emission, MS, and a variety of laser-based methods, is possible. The exploration of rfGD sources for analytical use has so far focused primarily on the development of rfGD-AES and rfGD-MS applications. The operating characteristics of any rfGD source (viz. its current, voltage and pressure) ultimately define the atomization-excitation-ionization characteristics. Briefly, discharge gas ions must be accelerated to the cathode surface and arrive with sufficient energy so that the collision cascade can produce a sputtering event (viz. atomization). The ion kinetic energy is thus affected by the discharge voltage (dc bias) and the source pressure, which affects the ion and atom mean free paths. Most of the discharge electrons are in fact created as secondary products in the sputtering process. Such electrons are accelerated from the cathode by the fall potential to the negative glow region. Within the negative flow, these electrons cause excitation of sputtered and discharge gas species as well as direct ionization of both. Long-lived (metastable) excited-state discharge gas atoms can in turn affect the ionization of sputtered species via a Penning-type process.

392

Devices for treatment prior to introduction into atomic ,spectrometers

Therefore, the extent of atomic excitation and ionization in this region is also dictated by the discharge power. As the application of rf powered GDs evolves, fundamental studies relating changes in operating parameters to the density and energy of sputtered atoms, electrons and ions, and metastable atoms, will become increasingly important with a view to obtaining better insights into analytical source design and operation. Comparison of the dc and r[operational modes in GC

Table 8.11 compares analytically relevant particle densities and energies in rf and dc GD devices. As can be seen, the values given for the two plasma types are fairly consistent. Therefore, one would expect that the analytical characteristics of rf and dc GD devices should be very different. A comparison of sputtering rates of metals for the rf atomic emission sources and conventional Grimm-type devices reveals no differences (0.1-1.5 mg/min). The key difference is of course the fact that the rf powered devices are capable of direct atomization of insulating materials. One other advantage, which has yet to be investigated, may result from the lower operating resources of rf devices producing a higher degree of depth resolution for thin film analyses. In practice, there exists a number of substantial differences in plasma energetics between dc and rf powered discharges. Comparisons of the two operating modes have been made with the same source simply by changing the power s u p p l i e s - in principle, any rf-designed discharge can operate in the dc mode, but the opposite is not necessarily true. Direct comparisons are shown in Table 8.11. The ion and electron densities, plasma potentials and electron energies were obtained with a Langmuir probe. The most salient differences are that rf plasmas are composed of electrons of much higher energy, whereas dc plasmas possess higher ion and electron number densities. Based on a simple Boltzmann analysis, these data suggest that rf plasma probably has more electrons of suitable energy for atomic excitation than does dc plasma and that high-lying excited states may be more extensively populated in the former. A number of mechanisms have been proposed to account for the enhanced electron energies in rfGDs [130], but the effect is most easily understood by picturing the 13.56 MHz. Additional energy is obtained by electrons when collisions with atoms give them a directionality in phase with the

TABLE 8.11 COMPARISON OF CHARGED PARTICLE CHARACTERISTICS IN THE SAME SOURCE Parameter

rf

de

Electron density, cm-3 Average electron energy, eV Excitation temperature, K Electron temperature, eV Ion number density, cm 3 Plasma potential, eV

2 • 101~ X 10l~ 4-7 5000-8000 1.5-2.5 3 • 101~ • 10I~ 9-16

6 x 10 I~ 18 x 101r 0.7-1.0 25 000-4000 0.2-0.6 4 x 10 t ~ 10l~ 2.4

Reproduced with permission of the Royal Society of Chemistry

Devices.[br treatment prior to introduction into atomic spectrometers

393

instantaneous field change. Electrons in a dc plasma feel no such polarity changes in the effectively field-free negative flow. While Wagatsuma and Suzuki [133] found no significant differences between the two operational modes of GD in 1997, Pan et al. found the following the next year: (a) an rf discharge usually exhibits higher analyte signal-to-background ratios than does a dc discharge, but the latter exhibits higher emission intensities; (b) an rf discharge becomes stable more quickly than a dc discharge and is more stable over an extended period, so the rf mode generally yields lower limits of detection; (c) the two modes provide the same between-sample reproducibility in terms of signal-to-background ratio, but the dc mode provides better such reproducibility in terms of emission intensity; and (d) a dc discharge sputters more rapidly than an rf discharge, but the latter exhibits much higher electron energies and hence higher excitation efficiencies [134].

8.3.3. Glow-discharge sources: geometry and improvements The first concern in designing a source with an optimal power is to ensure the maximum possible atomization-excitation-ionization capabilities for the applications at hand. Source geometry

The geometry of the discharge is critical in terms of isolating the bombarding species to the sample cathode and determining the energy of the bombarding ions as related to the dc bias in the case of rf sources. The first criterion is to maximize the ratio of the respective surface areas of the anode to the cathode. Only in highly asymmetric rf discharges is the dc bias located at the particular electrode. Specifically, ion bombardment is isolated to the smaller electrode (termed by analogy to dc sources as the cathode), with the negative bias approaching one-half of the rf peak-to-peak voltage in the ideal case. The actual distribution of electrode potential as related to both discharge symmetry and power coupling was reported by Kohler [ 135]. As shown in Fig. 8.12, six basic cases of symmetry and coupling can be described that expose the effect of geometry and mode of rf coupling (direct or capacitive) on both the dc bias voltage, Vdc (broken line), and plasma potential, Vp(t) (solid lines) a sinusoidal waveform was assumed. As illustrated, increasing the anode-to-cathode ratio increases the dc bias voltage and decreases the plasma potential. The distribution voltage is due to differences in the capacitances of the dark space sheaths of the two electrodes. At frequencies above a few MHz, the sheaths can be assumed to behave capacitively because the rf frequency is below the resonance frequency for the electron and above that for the ion. This means that an electron can cross the respective sheaths in less than one rf cycle, while a more massive ion experiences a number of rf oscillations. The glow-discharge can be adapted to many discharge types and ion source configurations. Occasionally, the sample type itself dictates a certain cathode geometry; other source models arise from more fundamental considerations. Most available glow-discharge sources can be assigned to one of the five simplified models depicted in Fig. 8.13. In each

394


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Fig. 8.12. Depictions of relative plasma potentials [Vp(t) (solid lines)], excitation electrode voltages [V(t), broken lines)] and dc bias voltages [Vl~.] as a function of the discharge coupling and geometry. (Reproduced with permission of the American Chemical Society.)

Devices./or treatment prior to introduction into atomic spectrometers

395

Fig. 8.13. Simplified representation of five common source configurations for glow-discharge. (Reproduced with permission of the American Chemical Society.)

396

Devices /br treatment prior to introduction into atomic spectrometers

case, ions are extracted from the negative glow of the plasma, which contains ions representative of the sample composition. A sampling orifice is located near the negative glow or a sampling cone is used to advantageously probe specific regions of the discharge. The most flexible source configuration is probably that of the pin source, which uses a spring clip or pin mount adaptable to samples ranging from thin wires to rods. Placed near the ion exit orifice, a small sample cathode (e.g. a 2 • 10 mm pin) at a low discharge current (1-5 mA) produces sufficient ion signal for sensitive elemental analyses. For sample materials that can be more readily cut into discs, the disc ion source may be preferable. Compared with the pin source, a somewhat different shape for negative glow is observed; however, equivalent current densities yield comparable signals from the two types of source. The hollow-cathode source provides one other form of glow-discharge [136]. It can be viewed as two planar cathodes brought close enough to each other to ensure that their negative glows will overlap and coalesce into a single common negative glow. This enhances cathodic sputtering and plasma ionization. Hollow-cathode ion sources provide excellent sensitivity; however, compared to pin or disc preparation, the machining of a hollow cathode from a bulk metal is occasionally more cumbersome. This configuration is particularly useful with samples in the form of metal foil or fragments that can be inserted into the cathode cavity for flow discharge. The hollow-cathode plume (HCP) [137] makes rather different use of the hollowcathode effect. The hollow-cathode base is a sample disc with a 1-2 mm orifice. By choosing appropriate gas pressures and discharge currents, the energetic plume is ejected through the orifice. A constricted hollow-cathode discharge localized in the narrow orifice creates intense sputter action on the surface of the disc and causes direct transport of the atoms into the plume for excitation and ionization, if required. The Grimm glow-discharge [138] is considered to be an obstructed discharge. By bringing the tubular anode within the cathode dark space, the discharge is obstructed or prevented from striking between the adjacent electrodes, thus restricting the discharge. This source can be used to analyse large samples without cutting or shaping of the material as the anode defines the sample area to be sputtered. I m p r o v e m e n t s in G D s o u r c e s

Glow-discharge sources, both dc and rf, have traditionally been operated in the continuous mode. The simplest original arrangement uses a low-cost supply with a discharge power often as low as 5-10 W; plasma species were thus formed with a steady-state stability of ca. 1-2%. Subsequently, advantages were gained by pulsing the discharge, boosting it using microwaves or magnetic fields, or employing secondary cathodes. Some commercially available power supplies are essentially power operational amplifiers that can be triggered by an external signal source. A pulse generator allows the power supply to be driven at selectable repetition rates and duty cycles. For a given average current, pulsed operation uses a higher voltage, higher current discharge during its "on" cycle than in dc operation. More energetic argon ions are produced, which enhances sputter yield and increases atom density in the plasma. Pulsed discharges also

Pulsed sources

Devices Jbr treatment prior to introduction into atomic spectrometepw

397

enable recording of time-resolved mass spectra by gating the detector at the desired interval along the pulse envelope. For example, more favourable ion signals from the sputtered species are obtained in the immediate post-pulse interval. A series of these pulsed sources have been reported that use various GD configurations; they were reviewed by Harrison [139], who subsequently conducted primary studies on a microsecond pulsed system based on a conventional hollow-cathode lamp [140] and on one based on the Grimm design [141]. In the latter, the authors investigated potential gains in signal-tobackground ratio (S/B) and limits of detection compared to a conventional dc operating mode [142]. They found the high instantaneous pulsed power itself to increase S/B by a factor of about 2 and obtained gains in the range 5-10. For nitrogen, they found that the background from leakage of air could be reduced to approximately one-third compared to the dc mode. One other interesting observation on the use of the pulsed mode was that some non-conducting materials that could not be run in the dc mode were successfully run in the microsecond pulsed mode. Discharge sources can be supplemented using various approaches that have been reviewed by Leis and Steers [143]. Magnetic enhancement of an ionization source for GD-MS was reported in the early 1980s [144], so its advantages and disadvantages have by now been clearly established. Results similar to those of a Grimm-type source [145]; much higher sputtering rates for insulating and conducting samples than an analogue source can provide without the field as a result of electrons being trapped in the discharge until they are deflected by collision [146]. Increased intensity in non-resonant emission lines for Cu(1) but decreased intensity in resonant ones are both the likely result of self-absorption [147]. Limits of detection 3--40 times lower in the presence of the magnetic field [148]; increased emission intensity ofAl, Cu and Ni (by 46%, 26% and 77%, respectively) [149]; and improved limits of detection ranging from 10 to 100 ng/g with ICP-MS [150] are a few other improvements afforded by GD-assisted methods with magnetic enhancement, which, however, require longer warm-up times and large arrays of sample types containing the analyte at concentrations over a wide range for calibration [151]. From the very beginning, the microwave-boosted glow-discharge lamp reported by Leis et al. [152] exhibited an enhancement factor of about 5 relative to a GD lamp; also, it diminished sample erosion but provided less uniform craters. Subsequent research on this type of lamp led to improved net emission intensities and S/B ratios, as well as to improved precision in the emission intensity and reduced self-adsorption in the glowdischarge [153]. A comparison of a glow-discharge, microwave-plasma and microwave plasma-enhanced glow-discharge with a steel sample as the cathode revealed differential distributions of the Ar spectral lines [154]. Because the intensities of the Ar(I) lines and OH bands were largely independent of the dc discharge current unlike the analytical lines, which depended strongly on it discrimination was achieved by modulating the dc current and using lock-in amplification, thus substantially reducing the interferences with the Bi(1) and AI(1) lines [155]. One straightforward, flexible form of microwaveboosted glow-discharge source is that based on a slab-line microwave cavity, which has been used to investigate fundamental excitation processes in unboosted and microwaveboosted GD sources [156]. The changes in spectral output can be described by the Boosted sources

398


TABLE 8.12 GENERAL EFFECTS OF THE SUPPLEMENTARY MICROWAVE DISCHARGE ON VARIOUS GROUPS OF LINES Emitting species

Group of lines

Typical values of F

Inert gas atoms

All lines

2-5

Inert gas ions

All lines

0.2-0.5

Atoms sputtered from the cathode

Lines from low lying upper levels Series of upper levels with same electron configuration n Upper levels close to ionization (above or below)

20-50 Falls with increasing n

Lines excited directly by charge transfer (CT) Lines excited by cascade from CT excited levels Lines not excited directly or indirectly by CT (upper level too high or too low, or CT prevented by selection rules)

0.1 0.4

Ions of cathode material

100-200

3


enhancement factors, F, of the different lines, F being the ratio of the intensity with the supplementary discharge to that without it, at constant current and pressure. Table 8.12 shows typical values for F. In addition to their expanding analytical applications, boosted GD sources can be of assistance in characterizing the excitation processes that occur in the discharge by examining the spectral changes involved [143]. Couples of cathodes of the same or different nature have been used for various purposes including the simultaneous comparison of a target sample and a reference standard - - thus avoiding the need for relative sensitivity factors - - [ 157]; correction of line interferences caused by the GD in AES (using double voltage modulation via the supplementary electrode) [158]; simultaneous AA and MS detection for comparison of atomic and ionic populations [159]; and, mainly, as secondary cathodes for the analysis of non-conducting samples using dcGD [ 160-163].

Dual cathodes

Jet-boosted GD in various configurations has been developed to improve precision, linearity and limits of detection in combination with AES [ 164]. Also, a two-cascade GD ion source proved highly effective with double-focusing MS, with which it provided sensitivity comparable to that of the ICP technique [165]. Finally, new hardware for rfGD from a Grimm-type excitation source enabled the analysis of very thin layers of target samples [166]. Other improvements"

Devices Jbr treatment prior to introduction into atomic .spectrometers

399

8.3.4. Nature of the sample for glow-discharge sampling Samples to be used in dc discharges must be electrically conducting. Metals, alloys and even semiconductors work quite well in this mode. Non-conducting samples must be mixed with a conducting matrix such as pure copper or silver and pressed into a suitable form. On the other hand, an rf discharge allows non-conducting samples to be handled directly, thereby avoiding the need for matrix modification. Radio frequency-powered GD sources have been coupled with various analytical spectrometries for more than two decades [167]. Analyses are not limited to conducting samples; in fact, electrical insulators such as glass, ceramics and geological materials some of the most difficult samples to dissolve for solution methods of elemental analysis can be readily examined.

8.3.5. Sample state and preparation for glow-discharge sampling Clearly, the principal use of GD is for solid analysis. At a time when many complex procedures are being examined for introducing solid samples into plasmas that are, by nature, better suited to solution injection, GD sputter sampling provides a simple procedure for conversion of a solid into an atomic vapour. Samples for dcGD solid sampling were originally mixed and compacted in conducting (metal) powders to form conducting sample discs for analysis. This matrix modification methodology has been used for many years in arc and spark emission spectrometries, and also in MS. Although relatively straightforward in implementation, this approach still poses a number of challenges as regards sample preparation (viz. grinding, drying and mixing) and the introduction of concomitant residual gases that affect source stability and analytical precision. These matrix modification steps are not required by rfGD analysis methods. The principal, distinct advantage of rfGD over traditional spectrochemical sources is its inherent capability for direct solid analysis of both conducting and non-conducting samples. Sample preparation for metals and alloys usually involves brief cleaning and/or polishing, although the sputter process itself serves as a self-cleaning mechanism for removing surface impurities. Powdered samples are compacted in a special die to form pins or discs. Special treatments (e.g. that for high-purity titanium, which is machined using an aluminium oxide cut-off wheel and double etched with a hydrogen peroxidebased acid etchant) reduce preparation times from 3 h to 20 min and pre-sputtering times from more than 1 h to 20 min [168]. Non-flat surface analysis requires special designs of both the anode and the cathode (where the sample is held) [169]. Samples from soil, rock or sediments are usually dried, homogenized and ground in a mill to a specific particle size (less than 50-100 jam) prior to analysis. They are thoroughly mixed with an amount of pure conducting matrix powder which is several times greater than the sample mass, and pressed into pellets, which produces dilution of the matrix. The conducting matrix usually consists of copper, but graphite, silver and tantalum have also been used for this purpose. The choice of copper as a binding material is particularly popular because the pressed pellet has good mechanical properties, excellent thermal and electrical conductivity and a high sputtering efficiency. However, this

400


approach is invariably limited by signal drift effects caused by preferential sputtering if the sputtering rates of the conducting host matrix and the non-conducting analyte material differ significantly. Samples obtained from animal or plant tissue must be processed prior to GD analysis. The sample may either be dried in an oven or ashed. The dry residue is homogenized and ground, after which it can be handled in the same manner as particulate matter. Solution analysis is also possible, but requires special manipulations. Volumes in the microlitre range are placed on a host electrode; as the solvent is removed, a thin-film sample residue remains that can be sputtered like any other solid. Alternatively, one can use the flow injection technique to insert small volumes of liquid samples. In any case, reduction of the dissolved sample into a thin surface residue allows very low limits of detection to be obtained from the transient signal. Marcus and Davis [170] recently used GD-AES at atmospheric pressure for direct sampling of liquid media, using an electrolytic solution containing the analyte specimen as one of the discharge electrodes. The passage of electrical current (either electrons or positive ions) across the solution/gas interface caused local heating and the volatilization of the analyte species. Collisions in the discharge region immediately above the solution surface resulted in optical emission that was characteristic of the analyte elements. The analyte solution acted as either the cathode or the anode. Preliminary limits of detection for Na, Fe and Pb were found to lie in the range 11-14 ppm for 5 ~tl sample volumes. Gases and volatilized samples can also be analysed with GD sources. Target analytes in a gas phase can be introduced in three different ways, namely: (a) by direct insertion of the gaseous sample; (b) by subjecting the sample to prior thermal vaporization; and (c) by using a chemical reaction (e.g. hydride generation or oxidation) to convert the analytes to volatile species prior to insertion into the GD source. In the last case, byproducts of the gas generation step such as water vapour, carbon dioxide and molecular hydrogen must be removed before they reach the discharge. A preconcentration step involving freezing of gaseous or volatilized samples on a cooled trap is often used prior to inserting the sample with a view to significantly improving limits of detection. One of the most salient applications in this context is the use of gas chromatography for the separation of gaseous or volatilized samples prior to introduction into the glow-discharge.

8.3.6. Variables affecting glow-discharge sampling The variables that influence performance in GD sampling can be classified according to whether they are shared by the source and detector or pertain exclusively to one or the other. The optimum value for each variable depends on the particular aim of the work at hand (e.g. quantitation in homogeneous samples, surface and depth profiling studies in layered samples). The most influential parameters in dcGD are the current intensity that circulates between the electrodes, the applied voltage and the pressure o f the plasma gas. In 1993, Payling and Jones devised a method for separating the contributions of the three key variables in these sources and proposed a quantitative approach in which the emission yield was assumed to depend on pressure alone [171]. This hypothesis was shown to hold for the rfGD mode as well [172].


401

The analytical performance of an rfGD for a specific sample is known to depend on three key operational parameters, namely: the pressure in the discharge chamber, the voltage between the electrodes and the applied power. However, owing to the interdependence between the three parameters, two of them can be fixed while the other is a free variable. The way the three discharge parameters affect the emission yield is not fully understood and is also a controversial issue. In the past, it was assumed that the emission yield depended on the discharge pressure only; today, however, it is accepted that the emission yield depends on the three key discharge parameters. Parker et al. [173] studied the role of the power and pressure parameters on the emission yield using rfGD and found slight changes in discharge pressure to affect it. On the other hand, Bengtson and H~instr6m [174] reported that the emission yield for a given spectral line reached similar values when the pressure was modified if the electrical parameters were kept constant. Recently, Sanz-Medel et al. studied the influence of the three operational modes (leaving the applied power, dc bias or Ar pressure as the free parameter) on the resulting qualitative and quantitative profiles giving information about the chemical composition of coatings and their thickness in a variety of samples. The results obtained in the three operating modes were consistent with those provided by dcGD-AES in a round-robin exercise conducted within the framework of a European Union project [175]; in any case, the worst relative standard deviations were obtained in the operating mode where the gas pressure was allowed to change [176]. Time-related variables are critical in GD work. One of the key features of any analytical method used in a production facility is the sample analysis time. Because GD is a kinetic-based system, the evolution of the plasma processes to, hopefully, an equilibrium situation, is of special concern. It is sufficient to say that any analysis (spectral acquisition) performed in a sequential mode requires that the plasma reach a steady state before analyses can be s t a r t e d - which is obviously arguable for simultaneous detection. Thus, for most analyses, the plasma stabilization time can be a substantial fraction of the overall sample analysis time. Short- and long-term plasma stability

The stability of a plasma source can be considered within two main time frames. First, there is a nominal period (short term) that is required for most simultaneous or sequential analyses and is 0.5-15 min in length. Short-term stability is also important when evaluating limits of detection based on the stability of the spectral background (RDSB), which is commonly used in emission spectrometry. The second level of stability (long term) applies to those instances where long integration times are used across a broad spectral region, taking upwards of 30 min which is typical of magnetic-sector MS. Gas type, pressure and flow-rate

These factors clearly influence plasma formation, the requirements for which depend on whether the GD source is of the rf or dc type and also on the detection technique to which the GD is coupled. As early as 1976, Gough [177] reported improved direct solid atomic absorption analysis achieved by using a flowing gas instead of a static cell and

402

Devices for treatment prior to introduction into atomic spectrometers 400

350 .__ E 300 :=L

1-

383

350

250

200 O -J 150 09

Id.I ._1 rl

343

100 5 ~ / . . 4355 2 2 50-

~3

-

cO

0 o

I

I

0.2

I

320 I

I

I

I

0.6

0.4 GAS

0.8

FLOW-RATE, I/min

Fig. 8.14. Average sample loss rate for a 6-min sputtering time versus gas flow-rate at three different current densities. The number below each point is the cell voltage: (Fl) 30 mA/cm-', (+) 40 mA/cm 2, (~) 60 mA/cm -~. (Reproduced with permission of the American Chemical Society.) correcting background absorption I which possibly resulted from aggregation of the sputtered atoms or particles with a dual-modulation amplifier. This contribution was greatly surpassed by the commercial six-jet Atomsource, which provided a higher sampling frequency in addition to proven enhancements in sensitivity and selectivity as shown by Kim and Piepmeier [178]. The influence of the gas flow-rate and current density on the sample loss rate I and hence on the amount of analyte available for adsorption n is illustrated in Fig. 8.14. Such influence together with that of pressure, power and orifice d i a m e t e r - has been confirmed with both conducting and non-conducting samples; in the case of rf sources, it is exerted not only on the sample loss rate, but also on the selfbias potential [179]. The direct effect of pressure on the atmospheric electrolyte-cathode GD on line intensity can be expressed using a mathematical model that also predicts the presence of an intensity peak [180]. The type of gas used to form the plasma is especially influential in some cases (particularly on line profiles and widths). Thus, operating a microwave-boosted GD source at 980 V at 30 mA with a microwave power of 40 W and Ne at 4.8 hPa as carrier gas prior to AES resulted in an erosion rate of 9 nm/s compared with 21 nm/s using similar conditions but Ar at 2.1 hPa as carrier gas [181]; the limits of detection for twelve metals in aluminium were similar with both, however. On the other hand, the type of gas employed dramatically affects the emission lines of halogens. Such is the case, for example, with the determination of fluorine using a Grimm-type GD source with Ne or Ar as the plasma gas. While detecting fluorine emission lines in an argon plasma is difficult, a neon plasma exposes not only the atomic lines but also the ionic lines for this element [182]. The excitation of fluorine may primarily be the result of collisional energy transfer from neon species having high internal energies. Conversely, use of the same source for sampling halogenated hydrocarbons in the determination of chlorine provided excellent perform-


403

ance when operated with helium [183]. In the case of alloys and a dc source coupled to AES, the use of pure Ar led to a strong dependence of the voltage-current characteristics of the discharge on the cathode material; on the other hand, a 2:23 Ar-He plasma mixture provided a discharge that was independent of the nature of the cathode [184]. Subsequent experiments with mixtures of these gases and an rf source also coupled to AES equipment revealed the measured electron and ion number densities, and the sputtering rate, to decrease with increase in the helium pressure but also to enhance the AI(1) and Cu(1) emission intensities, which was ascribed to measured increases in the average electron energies and electron temperature [185]. The spatial distribution of the analyte atoms is of paramount importance in atomic absorption techniques; in order to optimize atomic absorption signals, it is therefore desirable to find a region in the chamber where the concentration of sample atoms is maximal. In addition, the GD is an inhomogeneous medium, which makes knowledge of the spatial distribution of sputtered neutrals critical for AAS. One additional aspect to be considered in coupling a GD to an MS detector is ionization. Thus, given that the presence of oxygen or water vapour was known to adversely affect performance in dcGD-MS [186-188], the relative influence of Penning ionization on the relative sensitivity factors (RSFs) in dcGD as a function of the plasma gas was studied by using Ar, Kr and Xe. The latter two provided higher RSFs than Ar for the majority of elements studied [189]. When Kr was used as plasma gas, the addition of a small amount of H 2 (0.2%) reduced matrix effects compared to using Kr alone in the case of alloys [190] in MS. With non-conducting materials, the effect of small amounts of H 2, He, N 2 or 02 added to Ar increased the sputtering rate as a function of the concentration of the secondary gas, but with no effect on ion intensity [191]. One influential variable typical of rf sources is the aperture bias voltage, which has dramatic effects on the signal-to-noise ratio and has been extensively studied by Wagatsuma [192]; with a view to improving sensitivity, they controlled the voltage (either by introducing a new channel of dc current via a self-bias potential opened by using a low-pass filter circuit and a load resistor [193,194] or by applying a bias-current modulation at the frequency of the pulsed dc current) and achieved selective detection by using a lock-in amplifier at low noise levels [195]. One key parameter in the case of pulsed sources is the measurement gate, optimization of which dramatically improves the signal-to-noise ratio. The influence of other, minor variables can also be adjusted in order to improve performance. Thus, in addition to the typical parameters [viz. rf power, power density, plasma gas flow-rate and pressure, ion sampling distance, anode orifice diameter, and sample size, thickness and shape (pin-like or planar)], the efficiency of GD sampling as regards analytical stability, reproducibility, and quantitation and detection limits, also depends on cell configuration which has strong effects on radial diffusion processes. Self-absorption, which has a critical influence on AES sensitivity, could not be accurately quantified by using an equation relating the emission intensity of a resonance line affected by this phenomenon to the sputtering rate of the analyte concentration in the matrix [196]. Subsequent studies revealed that, for Cu and V, ionic lines are more sensitive than atomic lines - - the latter proved independent of self-absorption but were strongly influenced by the type of plasma used (Ar or Ne) [197].

404


8.3.7. Glow-discharge sampling as coupled to spectrometric detection Glow-discharge-mass spectrometry

Glow-discharge-mass spectrometry (GD-MS) has received increasing attention over the last decade. In fact, it is possibly the oldest form of MS. Electric discharges were commonly used as "natural" ion sources in the 1920s and 1930s for the early mass spectrographs [198]. Much of the early analytical work in GD-MS was aimed at replacing the spark ion source (a vacuum discharge ionization source that yields broad energy spreads, an erratic ion beam and usually unreliable quantitative results). The primary complicating factor in GD-MS source design is the need to accommodate the ion source volume within the main vacuum chamber. This allows cryogenic cooling of the discharge cell to remove trace impurities (which tend to decrease sputtering rates and result in the formation of molecular ions that increase spectral complexity) from the discharge gas. Thus, the sample must be transported to the cell volume through a vacuum interlock system by means of a direct insertion probe (DIP). The DIP design used in these assemblies is similar to those used in organic MS. The if-powered DIP must meet several design requirements including efficient coupling of the rf power to the sample, isolation of the sputtering to the sample surface and effective shielding of potential radiated power losses. The two designs in Fig. 8.15 are intended to afford great flexibility and allow the use of the same DIP for the actual sample insertion. Figure 8.15A is an expanded view of the pin-type sample holder implemented on the quadrupole instrument. This configuration allows direct mounting of the sample in the recessed region of the 8-ram diameter copper holder (mounted to the end of the rf feedthrough of the probe); the probe can thus be inserted into the discharge cell. Samples ranging from 1 to 5 mm in diameter can be analysed directly without excessive machining. This basic cell design has also been successfully implemented on the magnetic sector spectrometer. It affords easy sample changeover without breaking the source vacuum and provides for completely grounded coaxial protection of the powered rf lead. With samples less than 5 mm in diameter, the flat sample holder depicted in Fig. 8.15B affords direct analyses provided the sample is nominally flat. The holder is mounted to the end of the DIP and comprises a PTFE clamp assembly, support rods, boron nitride spacers and either stainless steel or aluminium anode orifice plates. The DIP is placed through the clamp assembly and the rf feedthrough is placed directly behind the sample (cathode), which locks it in place. The spacers separate the anode plate and the cathode in the same manner as the O-ring seal in the rf-AES source design. The anode body in this case is the Ta ion volume enclosed by the anode plate and the ion exit orifice mounted to it; the entire cell assembly is affixed to the commercial source cryo-cooling ring. The GD ion source gained popularity in the past two decades on account of its stability, sensitivity and operational simplicity. At least three commercial instruments were already available in the early 1990s. Elemental analysis by GD-MS provides substantial advantages over competing techniques in that it responds to metals and non-metals, exhibits a high sensitivity, suffers from minimal matrix effects and provides isotopic information. Unsurprisingly, it has developed into the most prominent GD coupling.


405

Fig. 8.15. Ion source designs for (A) pin and (B) flat sample types in rfGD-MS. (Reproduced with permission of the American Chemical Society.) One key difference between MS and optical detection techniques is that the sample material must be physically transferred out of the source. Analyte ions are extracted by electrostatic lenses and transferred through a mass analyser for eventual detection. Although ions are formed throughout the source cell, only those created very close to the exit orifice can survive to the high-collision environment and depart in the charged state. Both magnetic-sector and quadrupole-based instruments have been used in GD-MS, and commercial versions of each are available. The typical discharge operation conditions for GD-MS are 1-5 mA, 800-1500 V and 0.2-2.0 torr. Three-dimensional mathematical models of the behaviour of the different species in a dcGD in argon were used to study the influence of the dimensions of flat-cathode/ hollow-anode cylindrical cells on the calculated plasma quantities. The cell dimensions were found not to affect the plasma quantities qualitatively but only as regards their

406


B)

A) GAS

GAS

GAS

WATER TOR

PLANAR CATHODE j

WINDOW

WINDOW

NEGATIVE GLOW i)

L ANODE BODY WATER-COOL| CATHODE BO[

BODY INSULATOR GAS

ANODE WAT ER

GAS

- HOLLOW CATHODE

Fig. 8.16. Sources for GD-AES. (A) Grimm-type (planar cathode). (B) Hollow-cathode type. (Reproduced with permission of the American Chemical Society.) absolute values. Thus, for a discharge of 1 kV and 2 mA at 1 torr, the optimum cell for MS is one having 2 cm as both length and radius [199]. Glow-discharge-atomic emission

Glow-discharge-atomic emission techniques (GD-AE) for solid analysis are instrumentally simpler and more inexpensive than GD-MS [200]. Major trade-offs, however, include poorer limits of detection and reduced elemental coverage. Although compact commercial GD-AE instruments were popular in Europe during the 1980s, their use in the USA remained modest until the 1990s. Figure 8.16 depicts two commonly used versions of GD emission sources. The planar cathode or Grimm source in Fig. 8.16A is more common [ 138]. Also adapted for GD-MS applications, the Grimm source is an obstructed discharge in which the anode extends into the cathode dark space and defines the sample area that is sputtered. The sample is placed outside the source for easy access and changeover. Such a configuration is particularly adaptable to the analysis of metal sheets, discs or any fiat conducting sample that can be placed close to the source opening; powdered samples pressed into discs are also suitable for this purpose. The typical discharge operation conditions for a Grimm source are 500-1000 V, 25-100 mA and 1-5 torr. The planar cathode discharge is featured in commercial instruments designed for applications to trace elemental analyses in solids. Of special interest is its use for depth profiling of layered metal samples and for the analysis of geological materials such as granite, basalt, mica and feldspar without the thermal effects and self-absorption apparent in arc-spark operation.


407

The other, less commonly used source for GD-AE is the hollow-cathode discharge (HCD) shown in Fig. 8.16B, which features a cavity cathode rather than the planar cathode of the Grimm source [201]. The net effect of this difference at a given discharge voltage is concentration of the plasma within the constraining cavity, which enhances elemental emission intensities. Typical operating conditions are 200-500 V, 10-100 mA and 0.1-1.0 torr. For the analysis of metals and alloys, either the hollow cathode is machined from the bulk metal, or the sample (e.g. chips or powder) is placed in the hollow cavity of a pure material such as graphite. Solutions can be analysed by evaporating the sample to a residual film in the hollow cathode. Limits of detection usually range from 0.1 to 10 ppm. The coupling of HCD with microwave sources and the development of microcavities have helped improve the sensitivity of this type of source. Table 8.13 illustrates the excellent temporal features of rfGD-AES. Both metallic and non-conducting samples reach stability levels above 5% as RSD within an operating time of less than 30 s. In addition, analyte signals maintain a high degree of stability over single-element (1 min) and multi-element (5 min) analysis times. These temporal features suggest that the use of simultaneous detection is not required for bulk analyses (e.g. in arc and spark emission analyses). Analysis times could be more like those in arc and spark analyses (< 1 min) if multi-channel instruments were used. Table 8.13 also illustrates the external precision obtained for analyte concentrations at the tens of ppm level with both conducting and non-conducting samples. These values could be further improved by using internal standardization methods. Although GD-AES is generally considered to have good precision, the ability to obtain high levels of precision with the rf source when sputtering non-conducting samples is somewhat surprising. The effects of residual atmospheric oxygen and water vapour, which effectively quench the important metastable levels of the discharge gas atoms and inhibit the cathodic sputtering process, are well documented in the GD literature [202,203]. The sputtering of oxide samples such as glasses and ceramics most certainly introduces large amounts of oxygen into the plasma. Glasses and ceramics, which tend to be somewhat porous, also act as sources of water vapour that is released from the sample matrix in the sputtering process. Seemingly, however, the likely introduction of gaseous contaminants does not affect the basic operational stability or reproducibility of rfGD sources when operating in the AES mode. Although, based on Table 8.13, GD-AES possesses better analytical characteristics than GD-MS, in fairness, the development of analytical methods for MS is very easy compared to the labour-intensive selection of analytical transitions (wavelengths) required in AES. The identification of spectral interferences is also far easier in MS. Unfortunately, the number of instrumental plasma parameters that must be optimized results in a process that is much more complex than simply "making photons". Glow-discharge-atomic absorption

The glow-discharge is particularly suitable for atomic absorption (AA) techniques as the overwhelming majority of sputtered species are neutral atoms. This ability to produce a steady-state population directly from a solid matrix has obvious advantages that led Russell and Walsh to suggest the use of GD in the early development of AA [204].

TABLE 8.13 COMPARISON OF BASIC ANALYTICAL CHARACTERISTICS FOR rfGD-AES AND rfGD-MS Stabilization time to 5% RSD, min

Short-term'"] precision, YORSD

Long-term'"' precision, YORSD

External precision, YORSD

LOD

I

1

10-100 ppb

Method

Sample type

AES

Metals

0.5

0.05

AES

Nonconductors

0.5

0.8

MS

Pin-type metals

3

< I

1-100 ppb

MS

Pin-type nonconductors

30

I

8070-

o~

i--

I

COPPER CONCENTRATION, %

D),oo

1.4

~

(1 sample)

I I I I I I 0102030405060708090100

COPPER CONCENTRATION, %

~Cu

J

Cu:Fe (8 samples) . ~ / J y=0.0849x + 0.1038 ~ R2=09626

0.40.2

(~r I 80

Cu:Zn

12

ffl

7654-

C),,

1.4

/

~:

I 20

I 40

I 60

i.

.54._+.2.1nm

I 80

I 100

1 120

.

I 140

DEPTH, nm

Fig. 8.20. Quantitative depth analysis. (A) Plot of uncorrected calibrated data for various copper samples (Cu 219.227 nm emission) in different matrices. (B) Plot of sputtering rate-corrected copper calibration data of (A). (C) Time-intensity plot for a 50 nm layer of copper deposited on a steel substrate (intensities were measured using the Cu 219.227 nm and Fe 381.993 nm emission lines). (D) Normalized concentration-time plot from (C) for a 50 nm copper layer on a steel base. (E) Concentration-depth plot from (D) for a 50 nm copper layer on a steel base. (Reproduced with permission of the Royal Society of Chemistry.)

420

Devices Jot treatment prior to introduction into atomic spectrometers

method is not directly based on the emission yield concept. Rather, it relies on the proportionality between the sputtering rate and intensity. The essential difference from the IRSID method is that, in the SIMR method, the concentration rather than the sputtered mass of each element is used in the calibration function; this makes the SIMR method very similar to standard techniques for bulk analysis. Measurements are based on calibration at predetermined levels of voltage and current that are referred to as the "reference excitation conditions". One of the main goals of this method is to establish a calibration procedure resembling a standard bulk analytical method. Both quantitation methods are implemented with a steady-state dcGD. Pulsed GD introduces two additional parameters, viz. pulse width and pulse frequency, which provide further control over the sputtering process [243]. The first step in the quantitation method, once the sputtering rate has been determined and the instrument calibrated, involves collecting a raw timeintensity plot for a 50 nm layer of copper deposited on a steel substrate. The plot of Fig. 8.20C shows the variation of the intensity of the Cu(II) 219.227 nm and Fe 371.993 nm atom lines. The data in this plot only show intensity as a function of time, so they allow no qualitative information such as depth to be extracted. The next step involves determining the concentrations of the selected elements. This is done by converting the intensity data of Fig. 8.20B into concentrations, using the sputtering rate-corrected calibration curve for bulk standards shown in Fig. 8.20A. These concentrations are normalized to 100%. The results of the conversion are shown in Fig. 8.20D. The third step of the method involves determining the composite sputtering rate and composite density as a function of time from the normalized concentrations obtained in the previous step. The composite sputtering rate is simply the summation of the percent contribution of each element to the sputtering rate of the reference material over all the elements in the sample. Similarly, the composite density is the summation of the percent contribution of each element to the density of the reference material over all the elements in the sample. After the composite sputtering rates and densities have been calculated, they can be used in conjunction with the previously determined sputter area and sputter time to obtain the penetration depth (d). The sputtered depth increment, Ad, at a given point in time can be obtained by multiplying the penetration rate [eq. (2)] at such a point by the number of pulses per sampling point: Quantitation p r i n c i p l e

Ad, = w,p n

(8.4)

where Ad, is the sputtered depth increment (nm), w, the penetration rate (nm/pulse) at that point and p,, the number of pulses per sampling point. The overall penetration depth (d) can be obtained by summing all sputtered depth increments [eq. (4)] for each sampling point throughout the sampling range:

d- ~ ~a, t

0

(8.5)


421

Based on eqs (4) and (5), the time axis (s) of Fig. 8.20D can be converted to depth (nm) for the entire sampling depth (150 nm). Therefore, the final form of the data now reveals concentrations as a function of depth. Figure 8.20E shows the quantified result of the Cu layer on a steel substrate shown previously in Fig. 8.20C as a time-intensity plot. The experimental results obtained using the quantitative method predict a depth of 54 + 2.1 nm (1 s at the 95% confidence interval), consistent with the nominal depth of Cu reported for the sample. In addition, this plot provides more valuable information than do the time-intensity plots as it allows the concentration of the multiple layers to be monitored as the sample is being sputtered. Applications o f GD-MS

Glow-discharge coupled to mass spectrometry has been widely used for the analysis of metal alloys and high-purity materials. This is possibly the only GD-detector coupling that combines the required sub-parts-per-billion limits of detection with the breadth of coverage required for survey analysis. Glow-discharge mass spectra are much simpler than line-rich optical spectra, which makes qualitative analyses rapid and straightforward. Elemental ratio intensities are used for quantitation, usually with the matrix element acting as an internal standard. Polyatomic mass spectral interferences resulting from chemical combination of the discharge gas and the sample matrix (e.g. FeAr ~) can create problems at low concentration levels. Given the capabilities of modern detection systems, however, GD-MS ion intensities are linear over a wide range of concentrations, from major components to trace constituents. The advantages of GD-MS have turned it into a routine technique that provides reliable data on a daily basis in commercial laboratories. Quantitative analysis of such difficult elements as B, C, Si, and S in steels yields good results. The surface-active nature of GD ablation also enables the analysis of thin films and coatings. Although redeposition phenomena somehow limit the sharpness of layer resolution, GD-MS can be effectively used for such samples. The routine uses of GD-MS are rarely reported in the literature, which has led to an understatement of its actual usefulness. In any case, the maturity of this technique is quite apparent from examples such as the use of rfGD as interface with a 3D quadrupole ion-trap MS/MS instrument with efficiencies approaching 100% for oxides and hydroxides [246]; the low mass resolution for in-depth analysis of technical surface layers [237]; the time-resolved experiments involving ionized sputtered atoms in pulsed rf-powered GD-MS [247]; the design and characterization of an rfGD for double-focusing mass spectrometers providing representative spectra for glass, soil and brass matrices [248]; the development of new software for data processing of mass spectra acquired by high-resolution double-focusing GD-MS [249]; and the adaptation of a GD-MS system to a glove-box for the analysis of nuclear materials [250]. A comparison of atomization and ionization processes in dc, rf and microsecond pulsed discharges revealed the greatest mass loss to occur with the last type of source (two orders of magnitude greater than with the other two) [251]. Quantitative determinations have recently focused on high-purity materials [252]; trace noble metals (Pd, Pt and Th) in automobile catalysts [253]; and non-conducting [254,255] and semi-conducting materials [256]. The determination of 4~ in the presence of 4~

422

Devices fi~r treatment prior to introduction into atomic' spectrometers

as an illustration of the usefulness of time-gated detection [257]; the characterization of polymers [258]; and the use of solid-phase microextraction as a sample insertion technique for the analysis of organometallic compounds [259] are additional salient applications. Finally, surface analysis continues to be a common use of GD-MS [260]. Other applications of the GD-MS technique worth mentioning here include the monitoring of depth distribution of B and Li in ZrO 2 layers, where Zr alloy was corroded to ZrO 2 in an autoclave in solutions containing Li and B, samples being collected at different reaction times for analysis; the GD-MS crater depth and sputtering rate were determined profilometrically under the discharge conditions used [261]. The mechanisms of corrosion due to diffusion of impurities were also studied [262]. A comparison of dcGD-MS and ICP-MS for the analysis of trace elements in nuclear samples [263]; the development and evaluation of a GD-MIP source for TOF-MS [264] and a study of practical and theoretical aspects of GD-MS interfaces [265] are also worth mentioning here. Recent contributions have focused on coupling to gas chromatography. Such is the case with the dc gas sampling GD ionization source for the mass spectrometric analysis of halogenated hydrocarbons [266], where the continuous discharge is confined within the first vacuum stage of the differentially pumped spectrometer interface and can be either operated statically or rapidly switched between atomic and molecular ionization modes; both atomic and molecular spectra can be produced from a helium-supported plasma. Atomic and molecular limits of detection obtained with boxcar averager data collection were found to be comparable for static and switched operation (1-30 and 7-140 pg/s, respectively). The GC-rfGD-MS coupling has also been used in the analysis of organotin compounds [267]. Applications o f G D - A E S

Commercial GD-AES instruments designed for trace elemental analysis in solids use the planar cathode discharge [268]. Slightly different versions for correcting some deficiencies involving the use of a floating restrictor to decrease the curvature in the burning profile, Fourier transform (FT) for photon noise decrease, a boosting microwave discharge or a magnetic field are described in Section 8.3.3. Of special interest in this respect is the use for depth profiling of layered metals and the analysis of geological materials such as granite, basalt, mica and feldspar without the thermal effects and self-absorption apparent in arc-spark operation. For non-conducting samples such as airborne dust, an rfGD for the atomic emission determination of trace levels down to 2 pg/g of a number of elements in coal fly ash has been used [269]. One recent determinative application of Grimm-type GD-AES is the analysis for gold in precious alloys [270]. The hollow-cathode discharge, which relies on a cavity cathode rather than on the planar cathode of the Grimm source, is less frequently used. The net effect of this difference at a given discharge voltage is concentration of the plasma within the constraining cavity, which enhances elemental emission intensities. The performance of these lamps can be boosted with a view to lowering limits of detection and reducing interferences [271,272]. Most recent applications of GD-AES are concerned with surface analysis - - mainly depth p r o f i l i n g - of anodic alumina films [273], painted coatings [274], titanium carbide

Devices Jor treatment prior to introduction into atomic ~spectrometers

423

and nitride coatings [275] and electroplated ZnNi [276]. Additional applications include studies on the influence of amplitude modulation in depth profiling thin surface layers, the use of microsecond pulsed GD in quantitative depth analysis [278] and the examination of the influence of surface roughness on depth resolution [279]. Cho et al. [280] compared dc and rf gas jet-boosted GD-AES for the analysis of steel in terms of the voltage--current relationship; they studied the effects of the gas flow-rate (0-800 ml/min) and pressure (3-5 torr) on the dc bias potential, sample weight loss and emission intensity. The use of a simultaneous spectrometer for the rf mode proved dispensable by virtue of the high stability (variations less than 0.3%) for both matrix and trace elements. Both the rf and the dc mode provided calibration graphs that were linear over two or three decades (or even more if the analytical signal was normalized to the signal of a matrix component); however, the limits of detection obtained in the rf mode for many trace elements in steel were at the level of tens of ppb, which was an order of magnitude better than in the dc mode. GD-AES was found to surpass the energy dispersive X-ray (EDX) linescan technique in depth profiling capabilities; the former exhibited better turnaround time and spatial resolution, and required less sample preparation [281]. An additional, unusual application of rfGD-AES is the direct chemical analysis of bone [282]. GD-AES has also been used for GC detection in mercury speciation analyses [283]. Also, an atmospheric pressure GD atomic emission source was designed for direct sampling in liquid media; however, the preliminary limits of detection for elements such as Na, Fe and Pb were in the range 11-14 ppm (ca. 60 ng) for 5 pl sample volumes [284]. Specific applications to liquid and gaseous samples, and couplings to chromatographs for wet speciation analysis can be found in recent reviews such as that by Baude et al. [285]. Applications o f G D - A A S

Various types of GD have proved highly suitable for use as atom reservoirs for AAS; however, this solid sampling approach has been less frequently used in atomic absorption than in atomic emission and mass spectrometry, possibly as a result of the wider commercial availability of electrothermal devices. Studies of the effect of the discharge conditions [286] and those of the anode dimensions and location of the gas inlet port on the spatial distribution of the sputtered atoms [287] (both in an rfGD atomizer), and of the role of discharge parameters and the effect of limiting the orifice diameter [288], have been carried out. Additional applications of the GD-AAS couple include the evaluation of pulsed and transient atomization modes [289]; the use of a jet-enhanced sputtering device [290,291] and that of a thin-walled metallic hollow cathode [292]; the development of a new, two-component atomizer for the direct determination of medium and volatile elements in high-purity solid refractory metals [293]; and the use of an experimental design to optimize the analysis of nonconducting materials [294]. The glow-discharge also facilitates atomic absorption from non-ground state levels, which is often required in diode laser AAS.

424

Devices.fi~r treatment prior to introduction into atomic spectrometers

Applications o f G D - A F S

The glow-discharge is also highly suitable for use as an atom reservoir in fluorescence spectrometry as quenching of species through collisions is considerably reduced, which increases the populations of excited states in fluorescence work. Although conventional excitation sources such as an ordinary Xe arc lamp [295] - - which performs best in the pulsed mode [296] - - have also been used in this context, laser sources are far more commonplace here. For example, a miniature glow-discharge source assisted by laser in the excitation step was used by Winefordner et al. [205] for the determination of Pb with a limit of detection of 0.6 pg, and for those of europium, yttrium and thulium at the femtogram level [297]. Pulsed GDs have proved the most suitable for coupling to AF detectors [298,299] in most situations; however, a commercially available hollow-cathode lamp has also been used to approach single atom detection [300]. The GD-AFS coupling has also been used for in-depth resolution of microelectronic materials [301]. In any case, applications are still scant, especially if the high resolution and low limits of detection achieved so far are considered.

8.3.11. Trends in glow-discharge sampling Compared to other plasma sources such as ICE the glow-discharge has less stringent technical and financial demands as regards source construction and power supply. Most glow-discharges reported so far are operated at low pressures, so vacuum pumping is n e c e s s a r y - which is an obvious hindrance to the development of more affordable instrumentation. Two very recent developments in this context are the atmospheric electrolyte-as-cathode GD and the capillary GD, which overcome this shortcoming with liquid and gaseous samples, respectively. In the recently developed atmospheric electrolyte-as-cathode GD sources, a liquid serves as the cathode for the discharge, which facilitates direct analysis of continuously flowing liquids [302]. The device is potentially applicable to the continuous analysis for metals in water and wastewater. Using a different approach, Eijkel et al. [303] employed scaling theory to reduce electrode geometries by more than two orders of magnitude in comparison to the normal linear dimensions of a GD in order to generate a continuous glow-discharge in a capillary on a chip. They demonstrated the application as an emission detector for volatile organic compounds at either reduced or atmospheric pressures [304]. Although GD dimensions are extremely small, the microchip plasma can be successfully used for molecular emission detection; also, it exhibits limits of detection as low as 10-~4 g/s for methane and calibration curves that are linear over two orders of magnitude. Simple instrumentation, a small detector size and a high sensitivity make the device highly suitable for integration into microanalysis systems for volatile compounds, which could lead to the development of either new GD detectors or portable spectrometers for screening and field sampling. In relation to this application, GDs may become competitive with other plasma sources operated on a chip such as the microwave discharge developed by Engel et al. [305]. One other potential analytical use of GDs is as tunable plasma sources. Tunable sources can be operated in two modes to detect either atoms or molecules of (mainly) organic

Devices fi>r treatment prior to introduction into atomic ,wectrometers

425

compounds, depending on the conditions chosen for source operation. Details of various sources for this purpose are given in a special issue of the Journal o f Analytical Atomic Spectromet~ [306], which shows that they can be used for both emission and mass spectrometry. Normally, GDs are operated at a moderate or high power to ensure complete dissociation of molecules. When operated at a low power, the gas temperature is so low that soft ionization without molecular bond breakdown is possible. In emission spectroscopy, GDs can serve as detectors for elements such as C, N, O and H; in mass spectrometry, they can be used for the identification of molecules. They are thus theoretically suitable for direct (chemical) speciation analysis without the need for a prior separation. The most elegant feature of this approach is that, simply by changing the power, both operation modes can be realized. Glow-discharges can thus be used to solve analytical problems in areas of organic mass spectrometry where formerly only electron impact ionization, chemical ionization, fast atom bombardment, thermospray or electrospray ionization (ESI) could be used for soft ionization. Here, the borderline between inorganic and organic analysis is becoming increasingly blurred. Many innovative organic mass spectrometers equipped with ESI still have an additional atmospheric pressure ionization mode; they use a discharge that is highly similar to GD for enhanced detection of difficult to ionize organic compounds but they are mostly not operated in an atomic mode.

8.4. OTHER SOLID SAMPLING APPROACHES

A number of other devices are used for solid sampling, albeit to a much lesser extent than those discussed in the preceding sections. The most salient among them are described below. 8.4.1. Arc nebulization

Arc and spark sources for solid sampling are among the oldest described in the spectrometric literature. A relatively modern system of this type is the arc nebulization device developed by Jiang and Houk in 1986 [307] for the direct elemental and isotopic analysis of solid conducting materials (Fig. 8.21A). The sample was used as the cathode in an intermittent arc generated between a Cu anode and the sample cathode. The eroded sample material was transported through a 2 m long x 3 mm i.d. plastic tube into the torch of an ICP-MS instrument by an argon carrier gas flow. For the steel samples studied, the sample material was removed at a rate of ca. 1 mg/min. Standardization was done with steel SMRs the elemental composition of which was certified by NBS. Background spectra similar to those of LA-ICP-MS (see Section 9.2) were obtained that enabled the determination of non-metallic impurities. Limits of detection in the lower ~tg/g range were achieved for A1, Si, Ti, V, Cr, Co, Ni, As, Zr, Nb, Mo, Sn, Ta and W. The relative precision and accuracy were found to be ca. 5%. In a subsequent study, the arc nebulization system was used for the analysis of non-conducting ores and sediments [308], which were powdered, mixed with graphite and pressed into conducting pellets. Only small peaks for ArC t , ArO + and Ar2 t were detected in the background spectrum above

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m/z = 45. The small 0 2 peak observed at mass 32 enabled the determination of sulphur.

The limits of detection for the above-mentioned elements were 30-50 ~tg/g. Isotope ratios were determined with a precision of ca. 1% as RSD for the major constituents.

8.4.2. Direct sampling insertion devices At present, direct sampling insertion devices (DSID) exist for all atomic techniques. The earliest device of this type was developed by Horlick et al. for use with ICP-AES [309,310]. The system was subsequently refined to the final design shown in Fig. 8.21B. Thus, the Fassel-type torch was modified to accommodate the sample probe, which consisted either of laboratory-made graphite, tantalum or tungsten cups or of graphite sample cups used in classical dc arc emission spectroscopy. The cups sit on top of a quartz rod that is fixed on the sample cup holder. The bottom of the torch is sealed by a glass s h u t t e r - except during sample insertion so as to maintain the gas pressure in the central tube of the torch and keep the plasma discharge at its normal position. During insertion, the glass shutter is opened. When insertion is complete, a Teflon stop at the end of the quartz rods seals the bottom of the central tube. The sample can be dried or ashed at a position 35 mm below the load coil. When fully inserted, the top of the cup is aligned with the top of the coil. In this position, the sample cup can reach temperatures of 2000 K, but even at such levels, non-volatile analytes such as carbideforming elements do not vaporize completely. Sample size is of the order of a few milligrams (20 ~tl for liquid samples). This DSID is fully automated and features an overall time for full insertion of 5 s. This type of device can also be used with ICP-MS [311 ], where it provides increased sample insertion efficiency and much simpler background spectra in relation to ETV. In addition, matrix interferences can be reduced by removing the matrix at lower temperatures before the sample probe is fully inserted into the plasma [312,313]. The very small peak widths obtained during vaporization of the sample into the plasma can cause serious problems when the ICP-MS software or electronics is too slow for acquiring a sufficiently large number of data points. A recent, improved contribution is the use of the Rh-Cu couple for in-torch vaporization in direct elemental ICP-MS analyses in solid microsamples (sample mass ca. 130 lag), slurries and liquid samples (sample volumes in the microlitre range) [314].

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Chapter 9

Laser-assisted solid sampling 9.1. INTRODUCTION The time when physicists invented laser (about four decades ago, when some scientists referred to it as "a solution without a problem") seems very distant now. Today, lasers are everywhere around us (e.g. in bar code scanners, pointers, CD players, sophisticated lighting systems, etc.) in areas such as high-power plasmas for X-ray generation, pulsed laser deposition for thin-film coating, cutting and welding of workpieces with lasers, and tissue and bone surgery in laser medicine. Analytical chemistry has extensively exploited the advantages of laser by developing a wide variety of techniques that continue to grow in number day by day. In fact, the use of lasers in such a specific application as solid sample treatment is so wide, that this chapter has been limited in scope to direct analyses of the elemental composition of solids as a complement to Chapter 8. Hyphenated techniques involving time-of-flight (TOF) [1], matrix-assisted laser desorption ionization (MALDI) [2] or laser-enhanced Raman spectroscopy (SERS) [3] are not dealt with here because they are used mainly at the molecular level and, basically, in connection with liquid s a m p l e s - despite the fact that the sample becomes solid after the typical pretreatment applied. Also, those techniques used for direct desorption from natural solid samples (e.g. single-step and two-step laser desorption-ionization aerosol mass spectrometry) with a view to facilitating surface analysis are only considered in those cases where the laser acts on atoms (whether neutral or ionized), even if the final goal is a molecular determination (e.g. when metal ions are used for the chemical ionization of organic compounds and biologically relevant molecules [4-7]). Only the parent technique is described in detail, its derivations being illustrated with a few selected examples. This chapter thus deals with the use of lasers for removal of small portions of solid samples for subsequent elemental analysis. The physico-chemical form in which the material is removed, which dictates the nature of the subsequent steps of the analytical process, has given rise to different atomic techniques using previous laser sampling. Figure 9.1 shows the two principal groups of atomic techniques based on it. In one, known as "laser ablation" (LA), the sample is removed as an aerosol; no excitation of particles, atoms or ions is pursued in this step, but only the formation of sufficient small particles for transport to the atomizer, which can be a plasma source (particularly an inductively coupled plasma, ICP). In the ICP, the ablated material is heated at temperatures above 6000 K, which are high enough to cause breakdown and excitation and/or ionization of the atoms; subsequently, emission lines can be detected on deactivation of the target elements (LA-ICP-AES) or the species can be allowed to continue its path

436

Laser-assisted solid sampling Atomization/ ionization/

Detection

excitation

AES

'~v LA-ICP-AES

MS

~

LA-ICP-MS

.vAAS

~

LA-FI-AAS

"= ICP v

iv

LASER SOLID SAMPLING

vL

FLAME

ELECTROTHERMAL ATOMIZER

~

AES ~

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~I v

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Technique

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"~ LA-ETA-AAS

vL

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~ iv

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~,~

AES

'~v LIBS

~

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~

LIMS

Fig. 9.1. Techniques based on laser-assisted solid sampling/atomic detection.

through the interface and detected using a mass spectrometer. The combination is highly sensitive and limits of detection at the parts-per-billion or even lower are available for almost all elements in the periodic table. Only a small number of them (e.g. H, He, O and noble gases) are difficult to ionize and hence difficult to determine. When a mass spectrometer is used as detector, the combined approach is designated LA-ICP-MS. A flame, with subsequent absorption, emission or fluorescence monitoring (LA-F1-AAS, LA-FI-AES and LA-F1-AFS, respectively) or an electrothermal atomizer (LA-ETAAAS, LA-ETA-AES, LA-ETA-AFS) can also be used for laser ablation. Glow-discharge subsequent to LA is so scantily used that it does not warrant inclusion in Fig. 9.1; however, a selected example of its use is described among the applications. The detector connected in-line with the atomizer constitutes the last part of the assembled equipment and also the last acronym of the hyphenated technique. These techniques have the advantage that they separate the excitation step from the sampling step. As a result, both steps can be controlled and optimized separately. Most excitation steps are developed at atmospheric pressure, so no vacuum is required for sampling. The other group of atomic techniques based on laser sampling involves simpler couplings where only the detector is used after laser action the laser energy is used not only for ablation, but also for atomization, ionization and excitation, and a plasma is f o r m e d - and comprises "laser-induced breakdown" (LIB) or "laser-induced plasma" (LIP) techniques. The emission lines for most elements are plentiful and, because their widths are in the region of several picometres, a large enough number of lines usually exists for identification and quantitative analysis of most of the elements in the periodic table. The detectors used to monitor these lines are typically either normal or intensified charge coupled devices (CCDs); conventional diode array detectors have also been

Laser-assisted solid sampling

437

employed for this purpose, however. The excited species (ionized or not) can be either deactivated in situ (in laser-induced breakdown spectrometry, LIBS) or transferred to a mass spectrometer (in laser-induced breakdown-mass spectrometry, LIB-MS or LIMS). Techniques involving the use of two lasers (one for ablation and the other for atomization, ionization and excitation) are also discussed in this chapter. For a long time, both sampling approaches have been implemented by using a 1064 Nd:YAG laser providing 1 J pulses with a length of I ms. In addition to removing material, this laser was found to cause significant melting of samples, which was especially apparent from melt ejections in metals. As a result, the released material differed somewhat in composition from the sample: it contained more volatile than refractory elements by effect of fractionation. The use of UV lasers delivering high power densities (above 10 ~r W/cm-~), which are only available from pulsed lasers, suffices to cause non-thermal ablation and effectively restricts melting effects and fractionation.

9.2. LASER ABLATION Laser ablation is the more simple and popular group of the laser sampling techniques for elemental analysis. Also, it can be considered the first step of an LIB process. The ablation process encompasses erosion, melting, vaporization and sublimation. An ideal laser-based solid sample insertion system for any of the above-described atomizationionization-excitation sources should meet the following requirements: (a) it should require little surface preparation other than polishing and cleaning; (b) it should provide fine particles (no larger than a few microns in size); and (c) it should result in no selective vaporization and no matrix, memory or segregation effects. The extent to which a system fulfils these requirements depends on the properties of the laser (e.g. whether it is of the continuous wave or pulsed type, its wavelengths, its pulse duration and its repetition rate when pulsed). The type of analysis, either local or bulk, must also be considered. A local analysis can typically be accomplished by using a single-pulse or low-pulse rate laser. A transient signal will be obtained in both cases. Macro analyses require the use of a high pulse rate. Also, translation or rotation of the sample is needed to present a fresh surface to either each laser shot or set thereof. Finally, a steady state may be reached depending on the particular time constant of the transport configuration. 9.2.1. Features of ablation lasers

The performance of ablation lasers depends largely on two factors, namely: the nominal laser wavelength and intensity, and whether the continuous or pulsed mode of irradiation is used. Studies on the first two factors have yielded controversial results that can be ascribed to differences in sample nature or grain size. Thus, ruby (694 nm), Nd:YAG ( 1064, 532, 266 and, recently, 213 nm) and excimer (308, 248, 222, 193 and, recently, 157 nm) lasers have been used for laser ablation (LA). A comparison of the Nd:YAG laser operating at 1064 nm and 266 nm revealed better absorption of UV wavelength by most types of samples and also decreased fractionation. The particle sizes produced during ablation were smaller with 266 than with 1064 nm, which was possibly the reason for the reduced

438

Laser-assisted solid sampling SIDE VIEW

TOP VIEW 20 pm 30 pm 40 pm 60 pm 80 pm 120 pm

Fig. 9.2. Scheme of cascade ablation using crater sizes of 20 to 120 lam. (Reproduced with permission of Springer-Verlag.)

elemental fractionation observed at the former wavelength [8-10]. Experiments comparing the interaction of laser beams at 213 and 266 nm with solids [11] revealed improved ablation characteristics for highly transparent samples. In contrast to previous findings [12,13], non-sample related changes in signal intensities with time were found to be significantly smaller at the shorter wavelength. However, a recent comparison of laser beams at 266, 213 and 157 nm [13] revealed a similar extent of elemental fractionation with the three wavelengths. This suggests a decreased influence of wavelength relative to other factors such as energy density, focal point and the crater diameter to depth ratio. A comparison between a 266 nm Nd:YAG laser and a 193 nm excimer laser under closely matched conditions revealed improved ablation characteristics at 193 nm [13]. These results can be ascribed to differences in particle size distribution caused by the lasers. Little research, however, has been conducted at wavelengths below 266 nm. ICP-TOF-MS experiments confirmed the reduced elemental fractionation provided by a 193 nm ArF laser equipped with a homogenizing optical array. If elements are assumed to separate between particles according to volatility, a significant change in the elemental ratios between fractionating and non-fractionating elements will occur within the first few seconds of ablation for a stepwise enlarged ablation spot. The resulting ablation pattern is shown in Fig. 9.2 and the variation of the Sr/Zr, Ce/La, Pb/U, Pb/Th and Pb/Pb elemental ratios as a function of crater diameter in Fig. 9.3. Notwithstanding the increased pit sizes (20-120 ~tm), the elemental ratios obtained were very stable and no significant indication for element-selective redeposition on the sample surface was obtained. The Sr/Zr and Ce/La elemental ratios should not change as these elements are assumed to be refractory, so they should be very similar in terms of ablation behaviour. It should be noted, however, that the depth-to-diameter ratio of the ablation crater never exceeded unity. Ablation depths exceeding this aspect ratio are indicative of elemental fractionation, as reported by Horn et al. [14]. A relationship between fractionation and ionic radius, and one between charge and melting temperature, have been proposed. Fractionation also


439

Fig. 9.3. Elemental ratios measured for various crater sizes on the same ablation spot. Bars correspond to the following values from left to right: 20, 30, 40, 60, 80 and 120 lam. (Reproduced with permission of Springer-Verlag.) appears to correlate with the geochemical classification of elements (viz. lithophiles, siderophiles and chalcophiles) when using the 266 nm Nd:YAG laser to ablate geological samples [ 13]. Comparisons between laser wavelengths have been performed by using different samples under different output energies, with different optical arrangements, cell geometries and volumes. The results suggest improved ablation behaviour for highly transparent samples with lower (deep UV) wavelengths and less matrix-dependent ablation. In general, at any wavelength, the transparency of the sample influences the amount and extent by which it fractionates [14]. Recently, Gfinther et al. [15] conducted preliminary experiments using a 266 nm Nd:YAG laser ablation system with an optical assembly similar to that of the existing 193 nm unit for the purpose of examining the influence of wavelength (for equivalent optics) on the ablation characteristics. Experiments involving applying 3 mJ energy to the sample surface and the formation of a 100 ~tm crater resulted in the appearance of more transient signals than those observed at a low energy (0.5 m J); however, the calculated fractionation index [16] for the Pb/Ca ratio was still significant. In short, laser wavelength is not the sole critical parameter influencing fractionation as this phenomenon can be minimized or enhanced in any sample and at any wavelength depending on laser irradiance. An irradiance value can be established at which fractionation is either not significant or completely eliminated. No unequivocal relationship between an increase in signal intensity (viz. in the quantity of mass ablated and transported to the atomizer) and laser wavelength has so far been established. Lasers for solid sampling should always be operated in the pulsed mode. The continuous, CW mode, as often used for drilling and welding in engineering, would have serious drawbacks. The application of a CW laser provides much lower intensities than the peak intensities of pulsed laser. There would be continuous removal from the sample, which is closer to ordinary evaporation than the explosion-like ablation process of pulsed lasers.

440


The small area irradiated by a CW laser would continuously melt and the spatial composition in the liquid would change with time because of fractional evaporation and mixing in the liquid phase. Furthermore, the plasma above the sample would be continuously heated and, if the intensity is not low enough, it would shield the sample from the laser beam because of absorption by the plasma can be reduced by the choice of a laser with a shorter wavelength, a different kind of gas or a lower gas pressure. The most popular lasers for ablation have nanosecond pulse lengths. Sufficiently high intensities for explosion-like ablation can be used and there is additional heating of the plasma gas. The intensities, the wavelength, the kind of gas and its pressure, must, however, also be chosen carefully in order to avoid plasma shielding of the sample. The application of lasers with shorter pulses (e.g. in the picosecond or even femtosecond range) reduces heat dissipation by collisions of the high-energy electrons with the atoms in the solid, thereby minimizing melting of the laser crater region, as shown in Fig. 9.3, and fractional evaporation from the liquid phase. On the other hand, there is also less energy deposited in the gas plasma; this usually facilitates atomization of the material ablated from the sample. 9.2.2. Steps and thresholds in laser ablation

Steps of the laser ablation process Laser ablation involves two main steps, namely: laser-solid interaction and removal of ablated material.

Laser-solid interaction In this step, a laser beam impinges on a solid surface. Raising the temperature of the sample to a high enough level entails using a high laser energy. This can be accomplished by using a pulsed laser and ensuring accurate focusing of the beam. Properly focused ordinary lasers with pulses of a few nanoseconds and peak energies of several tens of a millijoule can provide energy densities (irradiance values) around 106-10 ~ W/cm 2. Under these conditions, the sample surface begins to absorb energy in the optical field, so its temperature increases gradually over the pulse duration. Separation of the material (ablation) When the temperature reaches a high enough level, particles separate from the sample surface through vaporization, boiling or sublimation and form a vapour phase between the solid and the laser beam, all at moderate irradiance values (below about 10~' W/cm2). The vapour thus formed is quite light and essentially transparent; with higher irradiance values, however, the vapour tends to absorb strongly at the beginning and can easily reach supersaturation. A significant amount of material can thus condense and form submicroscopic drops capable of scattering the incident laser beam. Thresholds in laser ablation There are at least two borderline situations as regards the interaction of a laser pulse with a surface in an ablation process. These situations are usually described quantitatively


441

as the lowest irradiance levels required to cause certain effects, namely: the damage threshold and the ablation threshold. The damage threshold is defined as the pulse irradiance required to cause a surface change that can be detected under the optical microscope. Although this definition is somewhat vague, it provides a useful irradiance limit that usually results in a detectable change in the optical properties of the sample surface. As a rule, any formation on the sample surface or its vicinity capable of inducing or enhancing the release of mass by the laser energy can start the damage and hence influence the damage threshold. Typical surface formations include electronic defects, impurities and particle inclusions. Laser absorption can be enhanced by fractures, voids and holes, grains, steps and various other irregularities. The ablation threshold is the irradiance value at which the material starts to separate from the sample. As a rule, the material separates through vaporization to a depth where the energy supplied by the laser beam equals the energy of vaporization. The material can separate in other forms including solid or liquid fragments and products of the photochemical decomposition of the sample. Because the energy deposited over the sample must exceed its latent heat of vaporization (H, J/kg), the threshold fluence (Fmin, J/m 2) is given by F"'i" :

pH ~a ~t--~

(9.1)

where p (kg/m -~) denotes sample density, a (me/s) thermal diffusivity and tc (s) pulse width. Heat effects considered, the ablation threshold thus depends on both the laser (for a given material) and the focal conditions (fluence). The ablated mass can be calculated from the expression M =-

E

(9.2)

C(T b - To) + H

where E is the laser energy per pulse (J), C is the specific heat (J/kg K), and Tb and To are the boiling temperature and initial ambient temperature (both in K), respectively. The sudden increase in crater depth observed during high irradiance (> 10 I~ W/cm e) laser ablation of silicon [17], which has been ascribed to phase explosion, can be used to establish a new threshold: the threshold irradiance for phase explosion. This threshold depends on two laser parameters, viz. beam spot size and wavelength. The larger the beam size and the longer the incident wavelength are, the higher is the laser irradiance required to cause phase explosion. 9.2.3. Craters and amount of ablated material

Based on existing results, the amount of material that is ablated from a sample ranges from 10 ng to 500 lag per pulse. The largest amounts (200-500 lag) are provided by freerunning ruby lasers; ruby lasers in the Q-switched mode provide less ablated material (typically 1 lag/pulse). Nd:YAG lasers give amounts in the 1 ng to 1 lag per pulse range, depending on their operating mode. In any case, these amounts of material are inadequate to match the sensitivity obtained with aqueous solutions.

442


Ruby and Nd:YAG lasers produce craters ranging from 1 to 500 ~tm in size. The craters are usually wider than deeper and exhibit overlapping rough materials at the edge suggestive of thermal effects. The rapid increase in crater depth above the threshold irradiance for phase explosion correlates with a significant increase in signal intensity. The ratio of crater volume to signal intensity, which represents the entrainment efficiency, remains the lowest at laser irradiances slightly above the phase explosion threshold. Such a ratio, however, increases at irradiances well above the threshold (> 10 II W/cm2). Particles differ in diameter and shape. Spherical particles are formed from molten material. Angular fragments are ejected directly from the surface. Dimensions can be up to 10 ~tm, which is too large to ensure efficient transport and thorough volatilization in the subsequent plasma, flame or electrothermal atomizer. The mass removal rate depends strongly on the laser power density, which is the main controllable parameter for a given material, and also on the laser wavelength when sampling solids. A wide variety of materials exhibit a decrease in the rate of change (rolloff) in the amount of mass removed with increasing incident laser power density. Roll-off results from a change in the efficiency of material removal by the laser beam and is primarily due to shielding of the target from the incident laser energy by a laser-vapour plume interaction. Studies on the amount of mass removed as a function of laser power density have revealed the change in ICP-AES intensity with laser power intensity to result from changes in mass removal efficiency. Roll-off in mass ablation is not due to a change in particle size distribution of the ablated species, fractionation of the sample or a change in transport efficiency to the ICP torch. Thus, accurate tracking of the laser ablation/ICP-AES process justifies the use of internal and external standards [18]. In the absence of matrix-matched standards, accuracy depends on the difference in degree of fractionation between the sample and standards.

9.2.4. Sample preparation for laser ablation Laser sampling allows a large number of samples to be processed without the need for preparation. This is not the case with every type of sample, however. The extent to which a sample requires preparation for laser sampling depends on its nature and on the particular information required. Valuable experience has been gained and reported for preparing solid samples for other solid analysis techniques; many of the ensuing procedures have proved extremely useful and convenient for laser sampling as well. In general, these procedures tend to be less labour-intensive than those required for solution sample preparation. With powdered samples, it can be of assistance to stabilize the sample, either by compacting it into a pellet (with or without a binding agent) or by fusing it to a borate glass. With non-homogeneous samples, it may be advisable to grind and mix the sample before stabilizing. A statistical study of the influence of sample inhomogeneity on the precision of analytical results was conducted by Scholze et al. [19], who applied a correlation function to the results obtained in the determination of selected elements in heterogeneous powdered soils. In order to obtain reliable information on the bulk composition of heterogeneous samples, a volume of about 20 mm 3 (equivalent to 50 craters ca.


443

1 mm deep each) was required for analysis. In this way, some analytical parameters such as the dependence of the expected relative standard deviation on the number of craters examined were determined. The minimum mass to be used was estimated from the number of craters to be obtained. The results were only semi-quantitative in most cases. In some special cases, quantitative analysis was possible provided the analyte was nearly uniformly distributed or the number of craters studied was increased in accordance. In some cases, it helps to either polish or etch the surface of a solid sample - - unless correlation between surface topography and composition is important. Small samples such as pins or crystals can be embedded in an epoxy cast and analysed either directly or after polishing the cast surface. Otherwise, small samples can simply be stuck into a piece of putty or onto tape or an adhesive pad. Because the sample cell is not evacuated, outgassing of the support material is not a problem with this approach. 9.2.5. Ablation cells

An efficient ablation cell must met several basic requirements, namely: (a) it should be easy to operate (particularly, it should allow easy sample changeover with some flexibility in sample size); (b) it should exhibit no memory effects; (c) it should allow the sample to be displaced in order to present a fresh surface to the laser action; and (d) it should possess a low dead volume. A wide variety of sample cell designs exists [20-24]; most use a flow of argon tangential, normal or concentric to the vapour plume to sweep vaporized material out of an enclosed sampling cell through the transfer tube. Because sample transport efficiencies are lower than unity, some sample redeposition can occur in the sampling cell and transfer tube. Usually, this causes no memory effects unless the material deposits on the cell window through which the laser beam is passed. Subsequent laser pulses can revaporize material previously deposited on the window, thus interfering with the analysis. This problem is avoided by systems with removable windows, which can be either replaced or cleaned. Ablation cell configurations can be classified into two broad categories depending on both the angle of laser incidence on the sample and the way the carrier gas is circulated through it, namely: (a) With focusing of the laser beam normal to the sample surface and the lateral inlet and outlet of the carrier gas (Fig. 9.4A). The flat sample can thus be rotated. This design is widely used, even though it is subject to some drawbacks such as a turbulent flow and material deposits on cell walls. The cell can be made of glass or a metallic material (stainless steel). Usually, the window can be easily replaced. In an early design [25], the sample was continuously brought by a tape. (b) With focusing of the laser beam either vertically downwards on the sample surface or with a given angle, up to a horizontal position (Fig. 9.4B) [26]. The carrier gas is injected into the cell. Usually, a tangential flow around the sample is used to provide a "sheathing" effect [21,26]. Material deposits onto walls are thus minimized. The carrier outlet is most often at the top of the cell [21,26]. One alternative design involves focusing of the laser beam on the lateral cylindrical surface of a

444


A)

B)

hv

l

/

Ar

[ . . . . . .

i1

hv

_

Ar

C)

TORCH SEMICONDUCTOR LASER

PIN HOLE

,//

,

i

PHOTOMULTIPLIER

t

10 mm

SAMPLE VAPOUR Fig. 9.4. Typical laser ablation cells with lateral inlet and outlet of the carrier gas (A) and tangential flow of the carrier gas (B). (C) Cross-sectional views of laser light-scattering cell.

rotating barrel with apparently inadequate ablation efficiency [27]. Other designs have been developed in order to improve the uptake rate [24], which constitutes a critical parameter. Specially shaped cells are required for special uses. One case in point is the cell depicted in Fig. 9.4C, which was designed to correct the variation of aerosol density in LA-ICP-MS and was a laboratory-made laser light-scattering cell for insertion into the aerosol transport tube between the laser ablation system and the torch. In this way, the temporal variation of signals from the laser light-scattering cell was correlated with that of the ion signals from the mass spectrometer [28].


445

Special cells are also designed for safety reasons such as avoiding contact with radioactive substances. Thus, based on other, previously reported designs [26,29], Leloup et al. [30] developed a sophisticated cell used in a glove box to reduce the volume of radioactive waste in the ablation of uranium samples. The ablation cell consisted of two main parts, namely: a mechanical, mobile part for sample positioning and a glass part for ensuring that the ablated material was carried away to the plasma torch, also located in a glove box. A more recently developed ablation cell (a jet-cell) directs the ablation gas flow of argon through a capillary onto the ablation side [31], which results in improved signals. However, the wide variety of potential target samples calls for different, specific ablation cells, so most laboratories using this technique employ two or more types of cell. A procedure for optimizing the geometry of these cells has been reported [32]. Most laser sampling systems use electronic stepper motors to move the stage on which the sample is mounted. Using a computer for remote control of the stage movement allows the sample to be moved precisely under the laser beam in various well-defined, reproducible translation programmes over a line or across a wide area, thus providing an alternative to single spot analysis. This approach is especially useful for analysing lateral compositional trends and for the bulk characterization of inhomogeneous materials.

9.2.6. Transport of ablated material Using a transport interface is almost mandatory in connecting the laser ablated material with the atomizer. There are few references to a direct connection to the torch [33]. Most workers use a tube made of PVC, Tygon or polyethylene with an i.d. of 2-7 mm and a length up to 150 cm. This results in large differences among time constants. In addition, deposits along the tube are also frequently observed. Few authors have examined transport efficiency in connection with ICP [34] and values as low as 5% at the torch level have been reported. A cyclone chamber can be used to remove exceedingly large particles [35]. Depending on the time constant and laser pulse frequency [36], a quasi-steady state can be achieved that allows the use of conventional data acquisition and processing systems.

9.2.7. Mixed gas sample introduction The ablation process has been studied under different gas environments to improve sample transport into the atomizer. Combinations of various gases (e.g. nitrogen, hydrogen and helium [37-41]) have been investigated. With ICP-MS, the use of nitrogen is leading to higher sensitivity in the high mass region, which is of especial importance for the determination of lead and uranium. Eggins et al. [39] achieved increased sensitivity (highmass region) by adding helium to the main flow of argon used to flush the ablation cell. The improved sensitivity thus obtained was mainly the result of decreased material deposition around the ablation crater. However, using pure helium (which was subsequently mixed with argon) as an ablation cell carrier gas resulted in increased sensitivity and reduced background intensities, thereby improving limits of detection [37]. The degree

446


of improvement was different for 266 and 193 nm laser systems. However, the source of this e n h a n c e m e n t - which might be related to particle size distribution or a change in the ICP conditions has not yet been quantified and requires further investigation. All mixed gas experiments (particularly those involving helium) alter gas dynamics at the interface, which makes it rather difficult to discard specific processes as the origin of the effects observed in alternative carrier gases for sample insertion.

9.2.8. Equipment Most laser ablation researchers design their own cells and transport systems, i.e. the laser-atomizer interface. Some routine users, however, purchase commercial equipment the characteristics and complexity of which depend on both the atomizer and the detection system. Laser ablation equipment is manufactured by CETAC Technologies for coupling with mass and emission detectors, under the names CSX-200 and Soils-500, respectively; they are the successors to the 1994 versions from CETAC. As stated by the vendor, the new systems are endowed with a flat-top beam profile, a geological package with a binocular viewing microscope, enhanced Windows TM software, unique laser energy control through innovative beam attenuation, pulse-repetition rate controlled through the laser Q-switch, user-friendly integration and improved stability. Also, the systems are so compact that they require no resonator alignment.

9.2.9. Calibration techniques for laser ablation-atomization-ionizationexcitation-detection The existence of reference materials and appropriate calibration procedures are two essential issues to be considered in quantifying the components of a sample. Quantitation has been substantially improved by the commercial availability of an increasing number of certified solid reference materials, especially for low concentration levels. Recently [42], NIST archival leaf standards were used as matrix-matched standards for reliable quantitative elemental analysis of Spanish moss samples. LA-ICP-MS was used with mixing standards in order to produce at least three data points for each calibration curve; the results thus obtained were compared with those provided by microwave digestion ICP-MS/AES. Standard addition was also examined and found to be an effective method in the absence of matrix-matched standards. An alternative calibration technique was developed and successfully applied to techniques hyphenated to LA [43--48]. In addition, an increasing number of matrixindependent calibration procedures using external reference materials and/or internal standardization procedures is being developed [49-51]. Although laser ablation usually dispenses with the need for sample preparation in the analysis of rocks, the use of a major element of the sample as internal standard requires the prior knowledge of its concentration; also, the addition of an internal standard element to a rock sample is difficult. Fusion of the sample with Li2B40 7 allows the use of Li and B as internal standards. Linear calibration graphs (constructed from standard reference geological materials) have thus been obtained for A1, Ca, Fe, K, Mg, Na, Si and Ti, and relative standard deviations of 1-2% (n = 12) achieved.


447

Several methods other than those based on internal standards have been tested with a view to improving precision in analytical methods involving laser ablation. Attenuation of a helium-neon laser beam due to scattering by ablated particles [52] and the sound waves generated in the sampling cell during the LA process [53] have been used to correct variations in the transport of sample vapour. The precision was increased by a factor of about 2 by normalizing the analyte emission intensities with the measured amplitude of the sound wave. Also, a laser light-scattering cell for measuring aerosol density was inserted into the transport tube of the sample vapour from the LA sampling cell to the ICP torch and the ion signals from the detector were normalized to the scattered light signals. Even in the single-pulse mode, the precision of the ion intensities normalized with the laser light-scattering signals could be improved to the same level achieved with the internal standard method [28]. Based on the results of their experiments with liquid samples in LA-ICP-MS, Gfinther et al. [54] developed a new approach to calibrating microanalyses of solids by this hyphenated technique. They used a liquid standard covered with a thin plastic film through which a small hole was drilled with the laser. Later, the introduction of liquid standards into the ablation cell by formation of either pneumatic [55] or ultrasonic aerosols [56] has allowed full exploitation of these approaches, which are illustrated in Figs 9.5A and 9.5B, respectively. Pneumatic aerosols require the use of a matrix element as internal standard owing to the inherent differences in sample insertion rate with LA and pneumatic nebulization. This poses problems with elements having no isotope with a relative abundance less than 0.1% as the resulting ion signals are too high for direct measurement and lowering the ablation rate compromises the sensitivity for trace elements. This shortcoming can be circumvented by using a high-stability ICP unit and mass filter, which allows the tail of a mass peak for the matrix element to be used as internal standard [55]. In calibrating the matrix element by nebulization with liquid standards, the ion signal measured on the peak tail is directly proportional to the element concentration in the ICE thus indicating that peak shape is not only stable, but also independent of peak height. The advantages of this method lie in the ease of preparing calibration standards for quantitative measurements with a laser ablation system and in access to homogeneous standard materials, which are difficult to homogenize in the solid state. Traces are calibrated relative to a fixed concentration of the matrix element. Calibrations thus performed for trace concentrations in high-purity copper provided good recoveries from high-purity reference standards. The ultrasonic nebulization-LA approach (Fig. 9.5B) was designed for multi-element determinations in geological samples; in order to match the two techniques, standard solutions were nebulized with an ultrasonic nebulizer (USN) during solution calibration and a blank target (e.g. lithium borate) was simultaneously ablated with a focused laser beam. Homogeneous geological samples were measured using the same experimental setup, where a 2% HNO 3 solution was nebulized simultaneously with the USN. Both homogeneous targets prepared from inhomogeneous geological samples by powdering, homogenizing and fusing with a lithium borate mixture in a muffle furnace at 1050~ and homogeneous geological glasses, were examined, and relative standard deviations in the range 2-10% (n = 6) were obtained for trace elements.


448 A)

LASER AEROSOL

NEBULIZER

L

MIXING GAS ADAPTOR

I

_

f

to ICP

SPRAY CHAMBER

B) AQUEOUS SOLUTION

to ICP-MS

ULTRASONIC NEBULIZER

Ar

ABLATION CELL

Fig. 9.5. (A) Schematic view of the combined use of laser ablation and pneumatic nebulization. (B) Experimental arrangement of LA-ICP-MS with solution calibration. (Reproduced with permission of Springer-Verlag.)

9.2.10. Shortcomings of laser ablation Some of the problems encountered in using the LA technique arise from so-called

elementalfi'actionation, a non-sample related change in the analyte response during the ablation process. This effect has been observed to some extent with all types of lasers, but other variables including energy density, focal point and the crater diameter-to-depth ratio also appear to exert a strong influence [9-13,39,51]. A number of specific approaches to minimizing elemental fractionation have been developed [11,14,54,57,58]. It remains impossible to pinpoint the source of fractionation:


449

ablation, transportation or excitation and ionization in the plasma, flame or electrothermal atomizer. Recent observations by Mank and Mason [12], and Russo [13], suggest that laser wavelength is not the dominant factor in elemental fractionation. The effects of the crater diameter-to-depth ratio appear to be more important. This has been confirmed by the results obtained with a 193 nm excimer laser ablation system for geochronological analysis [ 14]. Variations in laser irradiance constitute one other of the most serious shortcomings in LA. Fluctuations in the transversal mode structure and hot spots in the laser beam profile can give rise to significantly different ablation results. The beam profile should have at least Gaussian shape (TEM00), which produces "bell-shaped" craters. "Flat-top" beams, which have good homogeneous intensity distributions, are advantageous in laser sampling. They can be generated from Gaussian laser beams, either by expansion of the beam and use of an aperture which selects the central part or by application of specially designed optical arrangements of lenses or mirrors. Poor shot-to-shot reproducibility is one of the hindrances to using single-shot laser ablation (viz. one laser pulse per analysis) for the direct elemental analysis of solid samples with spatial resolution in addition to variations in the specimen being sampled. Proper data analysis appears to be one effective way of solving these problems. Two specific methods for improving singleshot measurement precision use the simultaneous full-spectrum acquisition capability of a time-of-flight mass spectrometer. In one, a normalization factor is computed from the combined signal yielded by all sample constituents, the reasoning being that the combined spectrum should be proportional to the total mass of sample ablated. This results in increased precision by a factor greater than 2, which is moderately better than is possible with a single internal standard. The increase in measurement precision is concentration-dependent, the greatest improvement (10-15 fold) being experienced by high-concentration elements. The other method correlates the attenuation of plasma matrix ions to analyte intensities. It provides no statistically significant improvement in precision, which is limited by the relatively low signal-to-noise ratio of the attenuated signals [59]. 9.2.11. Selected applications of laser ablation sampling prior to atomizationionization-excitation-detection Laser ablation is used in two major fields, namely: (a) bulk analysis, with a low spatial resolution (80-350 lam crater diameters) [60-63]; and (b) local analysis, with a high spatial resolution (4-80 gm crater diameters) [64-70]. Because laser ablation removes very small amounts of sample, a high sensitivity is essential for trace and ultratrace analysis. This makes the combination of LA with ICP-MS the most attractive and widespread choice. Depending on the particular application, virtually all types of mass filters including the quadrupole, magnetic sector field (with single or double focusing, single or multi-collector detectors) and time-of-flight types can be used in conjunction with LA; they have been found to improve the sequential and simultaneous determination capabilities of LA-ICP-MS and to arouse increased interest in this direct solid sampling technique as a result [71]. In fact, this powerful tool has been used for solid micro sampling analyses in geological [11,31,49,72-77], biological [78,79], environmental

450


[80-82], nuclear [30,83,84] and metallurgical samples [85]. It is an expeditious technique of relatively moderate cost that combines the high-sensitivity multi-element capabilities of ICP-MS and the advantages of in situ micro solid sampling by LA. It is particularly attractive to scientists wishing to study dissolution-resistant solid materials or spatial distributions of trace elements in a micro-scale area on the sample surface. However, the complexity of data acquisition and reduction of transient signals has so far restricted application of this technique owing to the need for continuous intervention of an operator. Recently, an automated method for the in situ trace element analysis of solid materials was reported [86] the performance of which was demonstrated by determining 22 trace elements in certified reference materials. The fact that laser ablation enables both bulk and local analysis has caused the demand for the flexibility of ICP mass spectrometers to rocket. The resolution of bulk analysis is typically 0.1-1 mm, whereas that of local analysis is usually between 5 and 50 ~tm; this results in a volume difference of 900 on the assumption of a constant ablation rate per pulse for a 10 ~tm crater relative to a 300 ~tm one. The linear dynamic range of the detector is therefore one of the most influential parameters for solid sampling. Major elements, usually employed as internal standards for quantitation, should ideally be measured together with minor and trace elements. Few commercially available systems are equipped with pulse counting and analogue mode amplifiers in the detection system resulting in 9-10 orders of magnitude in linear dynamic range. Analyte sensitivity as reported for different instruments is mainly influenced by the experimental crater size, energy density and repetition rate, which influence the amount of ablated and transported material. Normalizing sensitivities obtained with a similar energy density to a crater diameter of 40 ~tm and a repetition rate of 10 Hz leads to rather equal sensitivities of ca. 1000-3000 cps/ng for most commercially available instruments. The ensuing limits of detection are thus influenced mainly by background noise. One other critical parameter for multi-element analysis is the data acquisition rate, the effect of which has been extensively discussed by Longerich et al. [72]. Recent improvements in sector-field instruments ~ which have commonly been judged slow relative to magnet jump speed have been implemented in this context. Thus, measurement efficiency has been improved by using a short, fast change in the accelerator voltage for a magnet jump in such a way that the differences between quadrupole-based instruments and sector-field instruments have been brought to within a factor of 2 [87]. The ideal detection for short transient signals in laser ablation would be an "all-collecting" sectorfield instrument where a detector array would be located in the space and time focus collecting all isotopes fully simultaneously. The ICP-TOF-MS combination is near-ideal in this respect as all isotopes are sampled at once and detection is very fast [88,89]. The typical sampling time of an ICP-TOF-MS instrument is in the region of 5 las; also, with a read-out time of 12.75 ms (255 averaged spectra) the problem of spectral skew in sequential mass analyses is avoided. This provides access to elemental composition even for ultra-short transient signals and precise isotope ratios. The high data acquisition rate is required for the fast transient signals encountered, for example, during ablation of fluid inclusions.


451

Environmental applications

There has been sustained interest in the development of analytical methods for realtime monitoring of environmental pollutants in recent years. Semi-volatile organic compounds tend to be adsorbed onto the surface of aerosol particles of respirable sizes and pose potential health hazards. Conventional methods used for the analysis of organic compounds that are present on the surface of aerosol particles are based on particle collection followed by extraction and chromatographic analysis of extracted species [90,91]. These methods require large amounts of sample and long analysis times. Over the past decade, several research groups have reported real-time chemical analyses of aerosols [92-102]. Airborne particles are directly aspirated into a vacuum chamber where they are ablated and ionized by a laser pulse, followed by mass spectrometric detection of the ions. Several instruments are capable of providing size, number density and composition information about aerosols. The relatively high laser fluence required to ionize analyte molecules present on the surface of aerosol particles produces a high abundance of ions from the bulk material that can obscure weak signals from the surface-localized species. Charge-transfer processes specific to the matrix can also alter the analytical results [103]. Single-step laser desorption-ionization aerosol mass spectrometry is mainly suitable for the characterization of the chemical composition of the bulk material in the particles. Two-step laser desorption-ionization has been coupled with aerosol mass spectrometry to reduce fragmentation of organic species [104] or space charge defects in the ion source of the mass spectrometer [105], but none of the tests was specifically designed for analysing molecular adsorbates on particles. Recently, the surface analysis of particles by use of two-step laser desorption-ionization real-time aerosol mass spectrometry (L2RTA-MS) was accomplished [106]. The instrument used allowed real-time monitoring of polycyclic aromatic hydrocarbons (PAHs) adsorbed onto the surface of airborne particles without significant time delay between analysis and environmentally significant events. The one disadvantage of L2RTA-MS is that it requires two lasers, which complicates the equipment. This has led some authors to explore the use of real-time aerosol mass spectrometry (RTA-MS) for the detection of surfaceadsorbed species as an alkali metal adduct, thus taking advantage of the presence of easily ionized alkali metals contained in environmental particles. During laser desorption of surface-adsorbed analytes, Na ~ and K' ions are produced by the interaction of the laser radiation with the particle's material. The alkali metal ions serve as in situ chemical ionization reagents for the neutral analyte molecules. This new approach has been tested for monitoring tributyl phosphate (TBP) on the surface of environmental particles. The effect of laser fluence on the signal intensities of the potassium ion and cationized TBP was examined and the instrument found to perform best with laser fluences resulting in high abundances of K- but low abundances of ions from the particle's bulk material. The relatively low laser fluence needed to produce potassium ions avoided excessive fragmentation of the analyte. This approach allows real-time monitoring of sub-monolayer coverage of TBP on the surface of micro-sized particles [107]. Compared to detection by conventional L2-MS of aniline on silica surfaces as reported by Voumard et al. [108], the surface needed was at least 10~' times greater than that required by RTA-MS with in situ ionization. The greatest disadvantages of the latter

452


approach are fluctuations in the effective laser fluence - - on which the ion signal exhibits a steep dependence, especially in the region close to the ion production t h r e s h o l d - and the unexplored capabilities of MS/MS in this context (the charge of dissociated ions is carried by alkali metals, so no information about the organic compound can be obtained). On the other hand, the formation of adduct ions has the advantage that it shifts the m/z ratio for the pseudo-molecular ion to higher levels, where the background intensity is lower and few ions from the particle bulk interfere with detection of the analyte.

Biological applications The environmental monitoring of shellfish is a combined biological-environmental application of LA sampling; in fact, pollutants in seawater are incorporated into shells and can be used as indicators of environmental conditions. The hard parts of a shelly organism provide a time-based record of pollution. This additional information cannot be obtained with conventional soft tissue analyses. LA-ICP-MS as a direct solid sampling technique provides a spatial resolution of 150 ~tm with IR lasers, giving sub-parts-permillion limits of detection for carbonate materials [109]. Recent developments in laser technology can improve spatial resolution even further (to < 10 ~tm) by use of UV lasers, thereby enabling analyses between different growth bands of the shell and providing a way for establishing a timeline of pollution events. Such events may affect a whole population of shellfish, which can thus be used as a reference [110]. Determinations of metal components in biological materials provide responses to various problems, as shown by Cox et al. [111] in the trace element profiling of dental tissues. They used LA-ICP-MS to obtain elemental fingerprints for individual teeth, which were sectioned to produce a sample representing the time axis of tooth development. The ablated material was ca. 100 ~tm across, and the ablation process was reproducible and measured using phosphorus. The mercury and gold concentrations were found to decrease from the outer (mouth exposed) part of the tooth to the inner, newly deposited material. By contrast, the lead content was found to increase from the outer part to the inner part, thus reflecting the exogenous origin of the mercury and gold, and the endogenous origin of the lead from the blood supply. Determinations of the concentrations of other elements present in the teeth allowed the comparison of data between teeth through element-to-element ratios. The Ca/P ratio was recommended for this purpose. A more recent study based on a spatially resolved method enabled the determination of the migration of mercury from a filling into the tooth material and then into the human body, a process with obvious potential implications on human health. The interest in the use of other substances such as gold instead of amalgam for dental restoration was reinforced by the results [112]. The wide scope of LA-ICP-MS is reflected in its use for the characterization of annual growth rings in trees, which takes advantage of the fact that absorption at laser wavelength by a biological sample can differ from that of inorganic materials because the bonding energies of the molecules may be similar to photon energies and certain molecules can undergo photochemical changes. Therefore, in contrast to ablation of inorganic samples, organic material can show little or no evidence of melting or thermal damage. Calibration was done by using cellulose mixed with multi-element standard solutions and

64 nm 0.1-0.3 J/pulse

5.3 kPa

.

Sample holder

1.0 Vmin He

-

-

WATER

1mm

\

For powder samples

H

For sintered samples /

WATER Fig. 9.6. Schematic diagram of the experimental system for laser ablation-assisted radio-frequency atomization excitation. ( 1 ) Sample holder, (2) tantalum lid, (3) graphite cup, (4) graphite disc. ( 5 ) rf power source, (6) different types of sample holder for sintered ceramics, ( 7 ) Nd:YAG laser, (8) laser beam-focusing lens, (9) spectrometer, (10) quartz window for laser irradiation, ( I I ) central electrode, ( 1 2) discharge chamber, (13) quartz window for optical observation. (Reproduced with permission of the Royal Society of Chemistry.)

454


pressed into pellets. The elements studied (using a powerful Nd:YAG laser) included Mg, AI, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, Ba, TI, Pb, Bi and U. Although LA-based techniques provide only semi-quantitative results, the precision was high enough to allow the determination of elemental distribution and the identification of concentration trends [113]; this permitted the establishment of an autocorrelation function for the concentrations of elements in growth rings. The correlation time calculated from the autocorrelation function provided an indication of the transport of elements, thus allowing their migration from ring to ring to be studied [114]. One unusual application of LA-ICP-MS is for fingerprinting scene-of-crime evidence. The combination of this technique with ternary-plot pattern-recognition software was used to determine the origin of samples of steel (e.g. from safes, gun barrels, tools) and glass (e.g. floats, sheets, containers) [115]. A laser ablation-assisted glow-discharge excitation source with emission detection was used for the direct determination of elements in ceramics [116] using the experimental set-up depicted in Fig. 9.6. The sample was placed in a holder (1) that also served as hot electrode, and the atomizer consisted of a hollow Ta lid (2), a graphite cup (3) and a graphite bottom disc (4) with a cavity in which the powdered ceramic sample was loaded and irradiated with a Q-switch Nd:YAG laser (7) through a quartz window (10). Application of an rf power of 300-500 W at 13.56 MHz (5) to the central electrode (11) caused the formation of a stable He discharge plasma at a low pressure around the electrically conductive Ta hollow lid. Tantalum was also preferable for reaching higher temperatures because of lower radiative heat loss due to its low sensitivity relative to graphite. A different type of sample holder (6) was used for sintered ceramic samples in order to ensure a stable discharge, prevent the sample from melting with the Ta hollow lid and fix the sample location. Pulsed laser radiation of about 124 mJ per 10 ns was passed through the Ta hollow lid and successively hit the samples on the graphite disc at a rate of 10 pulses/s, resulting in increased atomization efficiency. The irradiation position of the sample surface was adjusted by focusing the lens (8). The atomic emission was detected by the spectrometer (9) and the analyte signal from the photomultiplier tube was acquired by a computer through an A/D interface. Either 5 lul of slurry or several milligrams of sintered silicon nitride was loaded in the cavity of the graphite bottom disc, the sample then being covered with the hollow lid and the sample cup fitted on top of the central electrode (11). The air in the discharge chamber was pumped out for 5 min to a pressure below 0.1 mm Hg and the helium was introduced into the chamber to attain a discharge pressure. Finally, the rf power was switched on and, concurrently, the computer started a programme to trigger the laser and acquire data in the boxcar integration mode. Effects of laser irradiation such as a sensitivity enhancement and alleviation of matrix effects were found in the direct atomization-excitation of ceramics. Figure 9.7 shows the marked enhancement of the emission signal for magnesium in the presence of laser sample irradiation and Table 9.1 shows the effect, in quantitative terms, of changes in the sample residue remaining in the sample holder, peak areas, relative standard deviations and normalized peak areas per ablated mass for A1. Peak area was increased by a factor about 4.9, which is greater than that obtained for a powder sample inserted into the system as a slurry. Also, the normalized peak area-to-ablated mass ratio for the ceramic material was increased by a factor of 2.0 and the reproducibility was improved.

Laser-assisted solid sampling A)

5 >.-

455

2400

1600 -

co z uJ z

800 -

i

B)

5

i

I

i

2400

1600 -

>.I

co z i.iJ Iz

800 -

0 0

I

I

I

I

4

8

12

16 TIME, s

Fig. 9.7. Enhancement of the glow-discharge emission signal for magnesium in the presence of laser irradiation (A) as compared with no laser assistance (B). (Reproduced with permission of the Royal Society of Chemistry.) Calibration using powder reference materials was possible for sintered ceramics. A very similar publication by the same authors, but in Japanese, was later reported [117]. Miscellaneous applications Although LA is too expensive to be the method of choice for solution analysis, the use of synthetic solutions and dual sample insertion systems for laser-induced aerosols mixed with liquids (dried and wet) has offset the lack of solid reference materials for quantitation [14,45-48,55]. A specific sample preparation procedure exists [118] for

456


TABLE 9.1 EFFECT OF LASER IRRADIATION ON A! EMISSION PEAK IN DIRECT EXCITATION OF POWDER CERAMICS (n = 4) Parameter

Without laser

With laser

Ratio

Average peak area (arbitrary units)

930 *

1600

2.6

RSD (%)

5.1

2.8

0.55

* Sample powderresidue was occasionally found in the cup. This value is based on 100% introduction Reproduced with permission of the Royal Society of Chemistry modifying the absorption of aqueous solutions used as calibration standards in the laser ablation of solids. However, the ablation behaviour of solutions is wavelength-dependent, in contrast to IR light, which is transmitted to a high degree in dilute solutions and therefore requires additives or modifiers to perform direct ablation analyses [45]. In a study involving the ablation of high matrix containing solutions (0.1 g Sn/l), samples were filled into polyethylene microtubes, sealed and placed in the ablation cell to minimize evaporation and cross-contamination. The laser was imaged onto the tube, and an 80 lam hole (2 mJ pulse energy, 10 Hz repetition rate) was ablated. The greatest difference between solid and liquid ablation was signal stability, which was poorer for liquids owing to droplet splashing during ablation. Quantitation was done by using identically prepared synthetic solutions for calibration. The accuracy of the determination of 1 lag/g and the related limits of detection were at the sub-ng/g level for most of the elements. A total volume of 11 lal of solution was analysed three times from the same tube, which led to absolute limits of detection of 2 pg. The greatest assets of this ablation procedure are its very low sample uptake, and the greatly reduced spectral interferences and matrix effects. In addition, this approach might be useful with a view to investigating spectral interferences in laser ablation.

9.2.12. Comparison of laser-assisted sampling with other sampling techniques Compared with many other techniques, sensitivity factors for LA-ICP hyphenated to either MS or AES are relatively uniform across the periodic table [21,22]. Also, the spectra for LA-assisted techniques are relatively simple and interferences between elements are more easily identified and corrected. ICP-AES was recently used in combination with HRICP-MS for the determination of 70 elements in coal using various sample treatments. Four microwave-assisted digestion procedures involving different dissolution mixtures (viz. nitric and hydrofluoric acids, aqua regia, hydrogen peroxide), lithium metaborate fusion with and without previous sample ashing, and direct sampling by laser ablation, were tested. The use of high-purity reagents, prevention from sample contamination and various correction procedures for spectral interferences provided limits of detection in the low nanogram-per-millilitre range for most of the elements studied. The precision was assessed from replicate analyses


457

(sample preparation included) of coal samples and found to average at 4-5% and 10-15% (as RSD) for procedures involving sample digestion and LA sampling, respectively. As regards dissolution mixtures, a combination of HNOJHF with H20 2 provided the best agreement with certified, recommended, literature-compiled and consensus values, even though quantitative recovery of Si, Cr, Hg, W, Zr and Y required the use of fusion. Overall, the results provided by LA fell within + 20% of those obtained by digestion [119]. The practical implementation of various spectroscopic analysis methods involving ICP-AES or LA-ICP-MS and two rather different types of sample was assessed and the following conclusions drawn from the study: (a) In the analysis of a magnetic material (NdFeB) by ICP-AES and LA-ICP-MS, accuracy in the determination of the major element (Fe) was found to depend on the particular dissolution method. Thus, Fe was preferably determined by classical titrimetry (see Table 9.2). However, the difference between the titrimetric and ICP results usually did not exceed 3%. When material other than Nd, Fe and B was to be examined, a survey analysis made use of a laser shot mandatory; the procedure revealed the presence of more than a dozen elements. Table 9.3 shows those that were subsequently determined by ICP-AES as well (following dissolution) to confirm the results. As can be seen from the table, the LA-ICP-MS results for the major elements were very poor but revealed at one glance the presence of substantial amounts of Co, Dy, Pr, Al, Si, Nb and Mo. (b) Sintered substances such as SrTiO~ posed dissolution problems. The question of whether a layer of SrTiO 3 deposited on a substrate of SrTiO 3 was possibly of lesser purity than the substrate itself was answered by using LA-ICP-MS (see Table 9.4). Subsequent neutron activation and ICP-AES analysis yielded the results also summarized in Table 9.4; prior dissolution of the powdered sample using an autoclave was required. These latter results confirmed the former. In fact, the initial LA-ICP-MS analysis provided all relevant information. The principal conclusion from the previous studies was that ICP-AES was the most rugged and flexible approach, but also that LA-ICP-MS was uniquely suitable for direct solid local analyses [120]. A comprehensive comparison of LA-ICP-MS with other techniques for the analysis of ceramic layers of solid oxide fuel cells was conducted by Becker et al. [121]. They TABLE 9.2 TYPICAL RESULTS OF ICP-AES ANALYSES OF THE MAGNETIC MATERIAL NdFeB Analyte

Concentration (% w/w)

Analyte

Concentration (% w/w)

Nd Fe" B Dy

30.4 66.0 1.01 1.49

Pr Ob Other elements Total

0.28 0.01 0.71 99.9

~'~Determined by titrimetry ~b~Determined by gas analysis


458 TABLE 9.3

ANALYTE CONTENTS (% m/m) IN AN NdFeB MAGNET AS DETERMINED BY LA-ICP-MS AND ICP-AES Ana lyte

L A-I C P-M S

IC P-AE S

Nd Fe B Co Dy Pr AI Si Pb Mo

52 40 0.16 2.0 3.4 1.3 0.50 0.28 0.50 0.092

30.1 59.5 1.06 3.15 2.00 0.70 0.80 0.12 0.095


TABLE 9.4 ANALYTE CONTENTS (gg/g) IN VARIOUS SrTiO 3 MATERIALS AS DETERMINED USING DIFFERENT METHODS

Analyte

Nd-doped SrTiO 3 target LA-ICP-MS

Ba Zr Nd Dy AI Cu Fe

110 15 4100 10 350 50 15

Nd-doped SrTiO 3 powder ICP-AES

Pure SrTiO 3 substrate

~'~ -- ~ - - ~''~ ~ ~'~ 306 73 11

25 < 1 < 1 < 1 < 20 < 5 < 5

--

LA-ICP-MS

NAA 17

< 5 < 1 < 3 < 1 : I-M

...........................................

LIBS 5 5 GW/cm 2

oo z w Fz

.

.

~149

i

0

25

-

I

i

50

75

LASER SHOTS Fig. 9.21. Emission profile of Zn (solid line) and Fe (broken line) in a zinc-coated steel studied by LIBS at two different laser irradiances and also by GD-AES. (Reproduced with permission of the Royal Society of Chemistry.)

492


9.3.12. Laser-induced breakdown-mass spectrometry The technique based on laser-induced breakdown coupled to mass detection, which should thus be designated LIB-MS, is better known as "laser plasma ionization mass spectrometry" (LI-MS). The earliest uses of the laser-mass spectrometry couple were reported in the late 1960s. Early work included the vaporization of graphite and coal for classifying coals, elemental analyses in metals, isotope ratio measurements and pyrolysis [192]. Later work extended these methods to biological samples, the development of the laser microprobe mass spectrometer, the formation of molecular ions from non-volatile organic salts and the many multi-photon techniques designed for (mainly) molecular analysis [ 192]. Figure 9.22 schematically depicts the experimental set-up used by Matus, Seufer and Jochum [193-198] for the direct analysis of rock surfaces [193], the microanalysis of mineral phases [194] and that of geological glasses [195] by LI-MS. These authors

Fig. 9.22. Laser plasma ion source. (Reproduced with permission of Springer-Verlag.)

Laser-assisted solid sanq~ling

493

developed a procedure for the quantitative determination of element concentrations [ 196] where the concentration of an element x was calculated from

C,=C, Hikb~:AW,: 9

1

a; AW, RSF,,

(9.3)

where C, is the concentration of an internal standard element; H;k is the measured ratio of the ion intensities of isotope i of element k and of isotope k of element s; a; and b~ are the abundances of isotopes i and k, respectively; AW, and AW~ are the atomic weights of elements x and s; and RSF,, is the sensitivity factor for element x relative to the internal standard element s [197]. Internal standardization in LI-MS results in improved precision and accuracy. An internal standard element corrects for potential interferences in ion yield and photoplate sensitivity. Geological glasses were analysed by using Ti as internal standard element, its concentration being determined by electron micropobe (EMP) and X-ray fluorescence (XRF) analysis. Relative sensitivity factors (RSFs) were used to correct for interferences in ion formation, transmission and detection of various elements. LI-MS exhibits only small differences in RSF because the high laser power density results in a uniform ionization efficiency [197,198]. Figure 9.23 shows the RSF values for 52 elements as obtained from analyses of certified reference materials. The experimentally determined RSF, r~ value ranged within 10% of that calculated from the following empirical formula:

=c; l, cR) 9 "fi

\/.ri, ]

\RTi

/ ,,., TTi/

(9.4)

where !, is the ionization energy (eV), R, the atomic radius and T, the boiling point (~ of element x. 10_

10

o

;..

- ,. '-,*...... I,,.

0.1

..'.,~

:2.

, "...r.: ........ ~ ..... "*'~"~"~';;".~.~..;.._.'_'_"~....,.'" 1

..._

llllllllllllllllllllllllllIlllllllIlllllllllllllllIl

K

Ti Mn Ni Ga

Ca Sc

V

Rb Zr

Fe Cu Ge Sr

Cr Co

Zn As

Ag Sn Cs Ce Sm Tb

Er

0.1 Lu W

Pb U

Nb Cd Sb Ba Pr Eu Dy Tm Hf Re Bi Y

Mo In

Te La Nd Gd Ho Yb Ta

TI Th

Fig. 9.23. Experimental values of the relative sensitivity factor for 52 elements from certified reference materials as compared with the theoretical values. (Reproduced with permission of SpringerVerlag.)


494 Pr

La

Ce 1000

Og iii n," s Z

o"iO

.~J-' i

100

Pm Nd

.

Sm

Eu Tb Ho Tm L u Gd Dy Er Yb

l_J_L ....

~

.

L._i

I

Ce

Pm Nd

: ~ , .... i

..t~_~._J

ATHO

Pr

La

Eu Tb Ho Tm Lu Gd Dy Er Yb i 1.1, i j__l i l._L__]: 1000

Sm

t i

STHS 6/80 L

t -4 -q.

"-...

.. '

:

i

9

100

O UJ

13_

]

00

10

1000 I ' ' .

III

n,' a Z O -1O TO

1 .

1 , - T - r - i i ~ r - r -]

" ; l~:--T-[,,,

~

"'

~

..........

TONALE

10 ]

1

1

1

1

I

!

I

1

r

1

r

I

1

1

1000

BCR-1G o..

~ J

100

B~;R-2G

III ._l

100

,

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